Methods for treating polymicrobial infections

ABSTRACT

Methods for detecting and treating polymicrobial infections, wherein a mixed population of microbes (e.g., bacteria) are present in a patient sample and the microbes are not first isolated from the sample. For example, the present invention describes specific polymicrobial infections and methods of treating said infections, wherein a particular antibiotic or a group of antibiotics are selected based on the composition of the polymicrobial infections.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a Divisional and claims benefit of U.S. patentapplication Ser. No. 16/848,651 filed Apr. 14, 2020, which is anon-provisional and claims benefit of U.S. Patent Application No.62/924,614 filed Oct. 22, 2019, U.S. Patent Application No. 62/928,815filed Oct. 31, 2019, U.S. Patent Application No. 62/956,923 filed Jan.3, 2020, U.S. Patent Application No. 62/977,637 filed Feb. 17, 2020,U.S. Patent Application No. 62/978,149 filed Feb. 18, 2020, U.S. PatentApplication No. 62/988,186 filed Mar. 11, 2020, and U.S. PatentApplication No. 63/009,337 filed Apr. 13, 2020, the specifications ofwhich are incorporated herein in their entirety by reference.

U.S. patent application Ser. No. 16/848,651 filed Apr. 14, 2020 is acontinuation-in-part and claims benefit of U.S. patent application Ser.No. 16/216,751 filed Dec. 11, 2018, which is a continuation and claimsbenefit of U.S. patent application Ser. No. 15/957,780 filed Apr. 19,2018 (now U.S. Pat. No. 10,160,991), the specifications of which areincorporated herein in their entirety by reference. U.S. patentapplication Ser. No. 15/957,780 is a non-provisional and claims benefitof U.S. Patent Application No. 62/487,395, filed Apr. 19, 2017, thespecifications of which are incorporated herein in their entirety byreference.

BACKGROUND OF THE INVENTION Field of the Invention

The present application is related to polymicrobial infections, moreparticularly to polymicrobial infections and therapeutic solutions fortreatment of said polymicrobial infections.

Background Art

Infectious diseases can affect multiple organs systems and areresponsible for significant morbidity, mortality, and economic impact.Infectious agents most often present as a complex polymicrobialinfections rather than as a single pathogen infection. Within the body,the pathogens of the polymicrobial infections coexist with each otherand through bacterial interactions change both the type of antibioticsthe organisms are susceptible and the level of antibiotics required totreat the infection.

It was surprisingly found that certain polymicrobial infections areassociated with changes in resistance, e.g., decreases in resistance orincreases in resistance, to particular antibiotics.

BRIEF SUMMARY OF THE INVENTION

Disclosed herein are methods for detecting polymicrobial infections,methods for treating polymicrobial infections, methods of guidingtreatment of a polymicrobial infection, etc., wherein a mixed populationof microbes (e.g., bacteria) are present in a patient sample and themicrobes are not first isolated from the sample.

The present invention describes specific polymicrobial infections andmethods of treating said infections by either killing the microbes ormethods of inhibiting growth of one or more of the microbes in apolymicrobial infection (e.g., inducing a bacteriostatic state), whereina particular antibiotic or a group of antibiotics are selected based onthe particular organisms in the polymicrobial infections. As isdescribed below, certain polymicrobial infections have a surprisingincrease or decrease in antibiotic resistance. For example, the presentinvention describes treating a polymicrobial infection with Klebsiellapneumoniae and coagulase-negative Staphylococcus (CoNS) withamoxicillin/clavulanate since it was surprisingly found that the apolymicrobial infection with both Klebsiella pneumoniae andcoagulase-negative Staphylococcus (CoNS) has a reduced odds ofresistance to amoxicillin/clavulanate.

The present invention also features methods for guiding the treatment ofparticular polymicrobial infections, e.g., methods for helping aphysician or other healthcare provider choose an appropriate antibioticfor treating a polymicrobial infection. For example, the methods featureproviding a physician or healthcare professional information (e.g., areport) about a polymicrobial infection, wherein the report providedincludes the odds of resistance the particular polymicrobial infectionhas to one or more antibiotics. The information can help guide thephysician in the decision-making process. Without this information, thephysician or healthcare provider may choose an inappropriate antibioticfor the infection, e.g., an antibiotic that the polymicrobial infectionhas increased odds of resistance to. Or, in certain embodiments, thephysician or healthcare may see that the polymicrobial infection has adecreased odds of resistance to a particular antibiotic that in the caseof a monomicrobial infection would not be appropriate for use. Or, incertain embodiments, the physician may see that the polymicrobialinfection is susceptible to a weaker antibiotic, whereas in the case ofa monomicrobial infection he/she may have opted for a strongerantibiotic. This may provide the physician with a broader range ofantibiotics from which to choose.

The methods described herein may feature administering an antimicrobialto the patient having or suspected of having a particular polymicrobialinfection. In certain embodiments, the methods herein also include thestep of detecting the presence of a particular combination of microbesin the polymicrobial infection, e.g., from a source of the infectionfrom the patient.

Klebsiella pneumoniae and Coagulase-Negative Staphylococcus (CONS)

As previously discussed, the present invention features a method oftreating a polymicrobial infection comprising Klebsiella pneumoniae andcoagulase-negative Staphylococcus (CONS) (or a method of killing orinhibiting growth of K. pneumoniae and coagulase-negative Staphylococcus(CONS)), wherein the method comprises introducingamoxicillin/clavulanate to the K. pneumoniae and CoNS polymicrobialinfection (e.g., administering amoxicillin/clavulanate to the patienthaving or suspected of having a polymicrobial infection with K.pneumoniae and CoNS), wherein K. pneumoniae and CoNS together have adecreased odds of resistance to amoxicillin/clavulanate.

The present invention also features a method of treating a polymicrobialinfection comprising K. pneumoniae and coagulase-negative Staphylococcus(CONS) (or a method of killing or inhibiting growth of K. pneumoniae andcoagulase-negative Staphylococcus (CONS)), wherein the method comprisesintroducing ceftriaxone to the K. pneumoniae and CoNS polymicrobialinfection (e.g., administering ceftriaxone to the patient having orsuspected of having a polymicrobial infection with K. pneumoniae andCoNS), wherein K. pneumoniae and CoNS together have a decreased odds ofresistance to ceftriaxone.

The present invention also features a method of treating a polymicrobialinfection comprising K. pneumoniae and coagulase-negative Staphylococcus(CoNS) (or a method of killing or inhibiting growth of K. pneumoniae andcoagulase-negative Staphylococcus (CONS)), wherein the method comprisesintroducing ciprofloxacin to the K. pneumoniae and CoNS polymicrobialinfection (e.g., administering ciprofloxacin to the patient having orsuspected of having a polymicrobial infection with K. pneumoniae andCoNS), wherein K. pneumoniae and CoNS together have a decreased odds ofresistance to ciprofloxacin.

The present invention also features a method of treating a polymicrobialinfection comprising K. pneumoniae and coagulase-negative Staphylococcus(CoNS) (or a method of killing or inhibiting growth of K. pneumoniae andcoagulase-negative Staphylococcus (CONS)), wherein the method comprisesintroducing levofloxacin to the K. pneumoniae and CoNS polymicrobialinfection (e.g., administering levofloxacin to the patient having orsuspected of having a polymicrobial infection with K. pneumoniae andCoNS), wherein K. pneumoniae and CoNS together have a decreased odds ofresistance to levofloxacin.

The present invention also features a method of treating a polymicrobialinfection comprising K. pneumoniae and coagulase-negative Staphylococcus(CONS) (or a method of killing or inhibiting growth of K. pneumoniae andcoagulase-negative Staphylococcus (CoNS)), wherein the method comprisesintroducing gentamicin to the K. pneumoniae and CoNS polymicrobialinfection (e.g., administering gentamicin to the patient having orsuspected of having a polymicrobial infection with K. pneumoniae andCoNS), wherein K. pneumoniae and CoNS together have a decreased odds ofresistance to gentamicin.

The present invention also features a method of treating a polymicrobialinfection comprising K. pneumoniae and coagulase-negative Staphylococcus(CONS) (or a method of killing or inhibiting growth of K. pneumoniae andcoagulase-negative Staphylococcus (CoNS)), wherein the method comprisesintroducing TMP/sulfamethoxazole to the K. pneumoniae and CoNSpolymicrobial infection (e.g., administering TMP/sulfamethoxazole to thepatient having or suspected of having a polymicrobial infection with K.pneumoniae and CoNS), wherein K. pneumoniae and CoNS together have adecreased odds of resistance to TMP/sulfamethoxazole.

Thus, the methods of the present invention also include treating apolymicrobial infection comprising K. pneumoniae and coagulase-negativeStaphylococcus (CoNS) may comprise introducing (e.g., administering) oneor more antimicrobials selected from: amoxicillin/clavulanate,ceftriaxone, ciprofloxacin, levofloxacin, gentamicin, orTMP/sulfamethoxazole.

The present invention also features a method of guiding treatment of apolymicrobial infection comprising Klebsiella pneumoniae andcoagulase-negative Staphylococcus (CoNS). The method may comprisedetecting the polymicrobial infection with K. pneumoniae and CoNS andproviding a report showing one or more antibiotics to which K.pneumoniae and CoNS have increased and/or decreases resistance to. Thereport may help the physician choose an appropriate antibiotic toadminister to the patient.

Coagulase-Negative Staphylococcus (CONS) and S. agalactiae

The present invention also features a method of treating a polymicrobialinfection comprising S. agalactiae and coagulase-negative Staphylococcus(CoNS) (or a method of killing or inhibiting growth of S. agalactiae andcoagulase-negative Staphylococcus (CoNS)), wherein the method comprisesintroducing ceftriaxone to the S. agalactiae and CoNS polymicrobialinfection (e.g., administering ceftriaxone to the patient having orsuspected of having a polymicrobial infection with S. agalactiae andCoNS), wherein S, agalactiae and CoNS together have a decreased odds ofresistance to ceftriaxone.

The present invention also features a method of treating a polymicrobialinfection comprising S. agalactiae and coagulase-negative Staphylococcus(CONS) (or a method of killing or inhibiting growth of S. agalactiae andcoagulase-negative Staphylococcus (CoNS)), wherein the method comprisesintroducing ciprofloxacin to the S. agalactiae and CoNS polymicrobialinfection (e.g., administering ciprofloxacin to the patient having orsuspected of having a polymicrobial infection with S. agalactiae andCoNS), wherein S. agalactiae and CoNS together have a decreased odds ofresistance to ciprofloxacin.

The present invention also features a method of treating a polymicrobialinfection comprising S. agalactiae and coagulase-negative Staphylococcus(CoNS) (or a method of killing or inhibiting growth of S. agalactiae andcoagulase-negative Staphylococcus (CoNS)), wherein the method comprisesintroducing levofloxacin to the S. agalactiae and CoNS polymicrobialinfection (e.g., administering levofloxacin to the patient having orsuspected of having a polymicrobial infection with S. agalactiae andCoNS), wherein S. agalactiae and CoNS together have a decreased odds ofresistance to levofloxacin.

The present invention also features a method of treating a polymicrobialinfection comprising S. agalactiae and coagulase-negative Staphylococcus(CoNS) (or a method of killing or inhibiting growth of S. agalactiae andcoagulase-negative Staphylococcus (CoNS)), wherein the method comprisesintroducing gentamicin to the S. agalactiae and CoNS polymicrobialinfection (e.g., administering gentamicin to the patient having orsuspected of having a polymicrobial infection with S. agalactiae andCoNS), wherein S. agalactiae and CoNS together have a decreased odds ofresistance to gentamicin.

The present invention also features a method of treating a polymicrobialinfection comprising S. agalactiae and coagulase-negative Staphylococcus(CoNS) (or a method of killing or inhibiting growth of S. agalactiae andcoagulase-negative Staphylococcus (CoNS)), wherein the method comprisesintroducing tetracycline to the S. agalactiae and CoNS polymicrobialinfection (e.g., administering tetracycline to the patient having orsuspected of having a polymicrobial infection with S. agalactiae andCoNS), wherein S. agalactiae and CoNS together have a decreased odds ofresistance to tetracycline.

The present invention also features a method of treating a polymicrobialinfection comprising S. agalactiae and coagulase-negative Staphylococcus(CoNS) (or a method of killing or inhibiting growth of S. agalactiae andcoagulase-negative Staphylococcus (CoNS)), wherein the method comprisesintroducing TMP/sulfamethoxazole to the S. agalactiae and CoNSpolymicrobial infection (e.g., administering TMP/sulfamethoxazole to thepatient having or suspected of having a polymicrobial infection with S.agalactiae and CoNS), wherein S. agalactiae and CoNS together have adecreased odds of resistance to TMP/sulfamethoxazole.

The present invention also features a method of treating a polymicrobialinfection comprising S. agalactiae and coagulase-negative Staphylococcus(CONS) (or a method of killing or inhibiting growth of S. agalactiae andcoagulase-negative Staphylococcus (CoNS)), wherein the method comprisesintroducing vancomycin to the S. agalactiae and CoNS polymicrobialinfection (e.g., administering vancomycin to the patient having orsuspected of having a polymicrobial infection with S. agalactiae andCoNS), wherein S. agalactiae and CoNS together have a decreased odds ofresistance to vancomycin.

Thus, the methods of the present invention also include treating apolymicrobial infection comprising S. agalactiae and coagulase-negativeStaphylococcus (CONS) may comprise introducing (e.g., administering) oneor more antimicrobials selected from: ceftriaxone, ciprofloxacin,levofloxacin, gentamicin, tetracycline, TMP/sulfamethoxazole, orvancomycin.

The present invention also features a method of guiding treatment of apolymicrobial infection comprising S. agalactiae and coagulase-negativeStaphylococcus (CONS). The method may comprise detecting thepolymicrobial infection with S. agalactiae and CoNS and providing areport showing one or more antibiotics to which S. agalactiae and CoNShave increased and/or decreases resistance to. The report may help thephysician choose an appropriate antibiotic to administer to the patient.

Coagulase-Negative Staphylococcus (CONS) and E. faecalis

The present invention also features a method of treating a polymicrobialinfection comprising E. faecalis and coagulase-negative Staphylococcus(CONS) (or a method of killing or inhibiting growth of E. faecalis andcoagulase-negative Staphylococcus (CONS)), wherein the method comprisesintroducing amoxicillin/clavulanate to the E. faecalis and CoNSpolymicrobial infection (e.g., administering amoxicillin/clavulanate tothe patient having or suspected of having a polymicrobial infection withE. faecalis and CONS), wherein E. faecalis and CoNS together have adecreased odds of resistance to amoxicillin/clavulanate.

The present invention also features a method of treating a polymicrobialinfection comprising E. faecalis and coagulase-negative Staphylococcus(CONS) (or a method of killing or inhibiting growth of E. faecalis andcoagulase-negative Staphylococcus (CoNS)), wherein the method comprisesintroducing gentamicin to the E. faecalis and CoNS polymicrobialinfection (e.g., administering gentamicin to the patient having orsuspected of having a polymicrobial infection with E. faecalis andCoNS), wherein E. faecalis and CoNS together have a decreased odds ofresistance to gentamicin.

The present invention also features a method of treating a polymicrobialinfection comprising E. faecalis and coagulase-negative Staphylococcus(CONS) (or a method of killing or inhibiting growth of E. faecalis andcoagulase-negative Staphylococcus (CONS)), wherein the method comprisesintroducing tetracycline to the E. faecalis and CoNS polymicrobialinfection (e.g., administering tetracycline to the patient having orsuspected of having a polymicrobial infection with E. faecalis andCoNS), wherein E. faecalis and CoNS together have a decreased odds ofresistance to tetracycline.

The present invention also features a method of treating a polymicrobialinfection comprising E. faecalis and coagulase-negative Staphylococcus(CONS) (or a method of killing or inhibiting growth of E. faecalis andcoagulase-negative Staphylococcus (CONS)), wherein the method comprisesintroducing vancomycin to the E. faecalis and CoNS polymicrobialinfection (e.g., administering vancomycin to the patient having orsuspected of having a polymicrobial infection with E. faecalis andCoNS), wherein E. faecalis and CoNS together have a decreased odds ofresistance to vancomycin.

Thus, the methods of the present invention also include treating apolymicrobial infection comprising E. faecalis and coagulase-negativeStaphylococcus (CONS) may comprise introducing (e.g., administering) oneor more antimicrobials selected from: amoxicillin/clavulanate,gentamicin, tetracycline, or vancomycin.

The present invention also features a method of guiding treatment of apolymicrobial infection comprising E. faecalis and coagulase-negativeStaphylococcus (CoNS). The method may comprise detecting thepolymicrobial infection with E. faecalis and CoNS and providing a reportshowing one or more antibiotics to which E. faecalis and CoNS haveincreased and/or decreases resistance to. The report may help thephysician choose an appropriate antibiotic to administer to the patient.

Coagulase-Negative Staphylococcus (CONS) and E. coli

The present invention also features a method of treating a polymicrobialinfection comprising Coagulase-negative Staphylococcus (CONS) and E.coli (or a method of killing or inhibiting growth of CoNS and E. coli),wherein the method comprises introducing amoxicillin/clavulanate to theCoNS and E. coli polymicrobial infection (e.g., administeringamoxicillin/clavulanate to the patient having or suspected of having apolymicrobial infection with CoNS and E. coli), wherein CoNS and E. colitogether have a decreased odds of resistance to amoxicillin/clavulanate.

The present invention also features a method of treating a polymicrobialinfection comprising Coagulase-negative Staphylococcus (CONS) and E.coli (or a method of killing or inhibiting growth of CoNS and E. coli),wherein the method comprises introducing ceftriaxone to the CoNS and E.coli polymicrobial infection (e.g., administering ceftriaxone to thepatient having or suspected of having a polymicrobial infection withCoNS and E. coli), wherein CoNS and E. coli together have a decreasedodds of resistance to ceftriaxone.

The present invention also features a method of treating a polymicrobialinfection comprising Coagulase-negative Staphylococcus (CONS) and E.coli (or a method of killing or inhibiting growth of CoNS and E. coli),wherein the method comprises introducing tetracycline to the CoNS and E.coli polymicrobial infection (e.g., administering tetracycline to thepatient having or suspected of having a polymicrobial infection withCoNS and E. coli), wherein CoNS and E. coli together have a decreasedodds of resistance to tetracycline.

The present invention also features a method of treating a polymicrobialinfection comprising Coagulase-negative Staphylococcus (CoNS) and E.coli (or a method of killing or inhibiting growth of CoNS and E. coli),wherein the method comprises introducing TMP/sulfamethoxazole to theCoNS and E. coli polymicrobial infection (e.g., administeringTMP/sulfamethoxazole to the patient having or suspected of having apolymicrobial infection with CoNS and E. coli), wherein CoNS and E. colitogether have a decreased odds of resistance to TMP/sulfamethoxazole.

Thus, the methods of the present invention also include treating apolymicrobial infection comprising CoNS and E. coli may compriseintroducing (e.g., administering) one or more antimicrobials selectedfrom: amoxicillin/clavulanate, ceftriaxone, tetracycline, orTMP/sulfamethoxazole.

The present invention also features a method of guiding treatment of apolymicrobial infection comprising E. coli and coagulase-negativeStaphylococcus (CoNS). The method may comprise detecting thepolymicrobial infection with E. coli and CoNS and providing a reportshowing one or more antibiotics to which E. coli and CoNS have increasedand/or decreases resistance to. The report may help the physician choosean appropriate antibiotic to administer to the patient.

E. faecalis and S. agalactiae

The present invention also features a method of treating a polymicrobialinfection comprising E. faecalis and S. agalactiae (or a method ofkilling or inhibiting growth of E. faecalis and S. agalactiae), whereinthe method comprises introducing ampicillin to the E. faecalis and S.agalactiae polymicrobial infection (e.g., administering ampicillin tothe patient having or suspected of having a polymicrobial infection withE. faecalis and S. agalactiae), wherein E. faecalis and S. agalactiaetogether have a decreased odds of resistance to ampicillin.

The present invention also features a method of treating a polymicrobialinfection comprising E. faecalis and S. agalactiae (or a method ofkilling or inhibiting growth of E. faecalis and S. agalactiae), whereinthe method comprises introducing vancomycin to the E. faecalis and S.agalactiae polymicrobial infection (e.g., administering vancomycin tothe patient having or suspected of having a polymicrobial infection withE. faecalis and S. agalactiae), wherein E. faecalis and S. agalactiaetogether have a decreased odds of resistance to vancomycin.

Thus, the methods of the present invention also include treating apolymicrobial infection comprising E. faecalis and S. agalactiae maycomprise introducing (e.g., administering) one or more antimicrobialsselected from: ampicillin or vancomycin.

The present invention also features a method of guiding treatment of apolymicrobial infection comprising E. faecalis and S. agalactiae. Themethod may comprise detecting the polymicrobial infection with E.faecalis and S. agalactiae and providing a report showing one or moreantibiotics to which E. faecalis and S. agalactiae have increased and/ordecreases resistance to. The report may help the physician choose anappropriate antibiotic to administer to the patient.

E. faecalis and P. miribilis

The present invention also features a method of treating a polymicrobialinfection comprising E. faecalis and P. miribilis (or a method ofkilling or inhibiting growth of E. faecalis and P. miribilis), whereinthe method comprises introducing meropenem to the E. faecalis and P.miribilis polymicrobial infection (e.g., administering meropenem to thepatient having or suspected of having a polymicrobial infection with E.faecalis and P. miribilis), wherein E. faecalis and P. miribilistogether have a decreased odds of resistance to meropenem.

The present invention also features a method of guiding treatment of apolymicrobial infection comprising E. faecalis and P. miribilis. Themethod may comprise detecting the polymicrobial infection with E.faecalis and P. miribilis and providing a report showing one or moreantibiotics to which E. faecalis and P. miribilis have increased and/ordecreases resistance to. The report may help the physician choose anappropriate antibiotic to administer to the patient.

E. faecalis and P. aeruginosa

The present invention also features a method of treating a polymicrobialinfection comprising E. faecalis and P. aeruginosa (or a method ofkilling or inhibiting growth of E. faecalis and P. aeruginosa), whereinthe method comprises introducing meropenem to the E. faecalis and P.aeruginosa polymicrobial infection (e.g., administering meropenem to thepatient having or suspected of having a polymicrobial infection with E.faecalis and P. aeruginosa), wherein E. faecalis and P. aeruginosatogether have a decreased odds of resistance to meropenem.

The present invention also features a method of guiding treatment of apolymicrobial infection comprising E. faecalis and P. aeruginosa. Themethod may comprise detecting the polymicrobial infection with E.faecalis and P. aeruginosa and providing a report showing one or moreantibiotics to which E. faecalis and P. aeruginosa have increased and/ordecreases resistance to. The report may help the physician choose anappropriate antibiotic to administer to the patient.

E. faecalis and K. pneumoniae

The present invention also features a method of treating a polymicrobialinfection comprising E. faecalis and K. pneumoniae (or a method ofkilling or inhibiting growth of E. faecalis and K. pneumoniae), whereinthe method comprises introducing meropenem to the E. faecalis and K.pneumoniae polymicrobial infection (e.g., administering meropenem to thepatient having or suspected of having a polymicrobial infection with E.faecalis and K. pneumoniae), wherein E. faecalis and K. pneumoniaetogether have a decreased odds of resistance to meropenem.

The present invention also features a method of treating a polymicrobialinfection comprising E. faecalis and K. pneumoniae (or a method ofkilling or inhibiting growth of E. faecalis and K. pneumoniae), whereinthe method comprises introducing tetracycline to the E. faecalis and K.pneumoniae polymicrobial infection (e.g., administering tetracycline tothe patient having or suspected of having a polymicrobial infection withE. faecalis and K. pneumoniae), wherein E. faecalis and K. pneumoniaetogether have a decreased odds of resistance to tetracycline.

Thus, the methods of the present invention also include treating apolymicrobial infection comprising E. faecalis and K. pneumoniae maycomprise introducing (e.g., administering) one or more antimicrobialsselected from: meropenem or tetracycline.

The present invention also features a method of guiding treatment of apolymicrobial infection comprising E. faecalis and K. pneumoniae. Themethod may comprise detecting the polymicrobial infection with E.faecalis and K. pneumoniae and providing a report showing one or moreantibiotics to which E. faecalis and K. pneumoniae have increased and/ordecreases resistance to. The report may help the physician choose anappropriate antibiotic to administer to the patient.

E. faecalis and E. coli

The present invention also features a method of treating a polymicrobialinfection comprising E. faecalis and E. coli (or a method of killing orinhibiting growth of E. faecalis and E. coli), wherein the methodcomprises introducing ampicillin/clavulanate to the E. faecalis and E.coli polymicrobial infection (e.g., administering ampicillin/clavulanateto the patient having or suspected of having a polymicrobial infectionwith E. faecalis and E. coli), wherein E. faecalis and E. coli togetherhave a decreased odds of resistance to ampicillin/clavulanate.

The present invention also features a method of treating a polymicrobialinfection comprising E. faecalis and E. coli (or a method of killing orinhibiting growth of E. faecalis and E. coli), wherein the methodcomprises introducing ampicillin/sulbactam to the E. faecalis and E.coli polymicrobial infection (e.g., administering ampicillin/sulbactamto the patient having or suspected of having a polymicrobial infectionwith E. faecalis and E. coli), wherein E. faecalis and E. coli togetherhave a decreased odds of resistance to ampicillin/sulbactam.

The present invention also features a method of treating a polymicrobialinfection comprising E. faecalis and E. coli (or a method of killing orinhibiting growth of E. faecalis and E. coli), wherein the methodcomprises introducing levofloxacin to the E. faecalis and E. colipolymicrobial infection (e.g., administering levofloxacin to the patienthaving or suspected of having a polymicrobial infection with E. faecalisand E. coli), wherein E. faecalis and E. coli together have a decreasedodds of resistance to levofloxacin.

The present invention also features a method of treating a polymicrobialinfection comprising E. faecalis and E. coli (or a method of killing orinhibiting growth of E. faecalis and E. coli), wherein the methodcomprises introducing meropenem to the E. faecalis and E. colipolymicrobial infection (e.g., administering meropenem to the patienthaving or suspected of having a polymicrobial infection with E. faecalisand E. coli), wherein E. faecalis and E. coli together have a decreasedodds of resistance to meropenem.

The present invention also features a method of treating a polymicrobialinfection comprising E. faecalis and E. coli (or a method of killing orinhibiting growth of E. faecalis and E. coli), wherein the methodcomprises introducing tetracycline to the E. faecalis and E. colipolymicrobial infection (e.g., administering tetracycline to the patienthaving or suspected of having a polymicrobial infection with E. faecalisand E. coli), wherein E. faecalis and E. coli together have a decreasedodds of resistance to tetracycline.

Thus, the methods of the present invention also include treating apolymicrobial infection comprising E. faecalis and E. coli may compriseintroducing (e.g., administering) one or more antimicrobials selectedfrom: ampicillin/clavulanate, ampicillin/sulbactam, levofloxacin,meropenem, or tetracycline.

The present invention also features a method of guiding treatment of apolymicrobial infection comprising E. faecalis and E. coli. The methodmay comprise detecting the polymicrobial infection with E. faecalis andE. coli and providing a report showing one or more antibiotics to whichE. faecalis and E. coli have increased and/or decreases resistance to.The report may help the physician choose an appropriate antibiotic toadminister to the patient.

E. coli and S. agalactiae

The present invention also features a method of treating a polymicrobialinfection comprising E. coli and S. agalactiae (or a method of killingor inhibiting growth of E. coli and S. agalactiae), wherein the methodcomprises introducing ampicillin to the E. coli and S. agalactiaepolymicrobial infection (e.g., administering ampicillin to the patienthaving or suspected of having a polymicrobial infection with E. coli andS. agalactiae), wherein E. coli and S. agalactiae together have adecreased odds of resistance to ampicillin.

The present invention also features a method of treating a polymicrobialinfection comprising E. coli and S. agalactiae (or a method of killingor inhibiting growth of E. coli and S. agalactiae), wherein the methodcomprises introducing cefepime to the E. coli and S. agalactiaepolymicrobial infection (e.g., administering cefepime to the patienthaving or suspected of having a polymicrobial infection with E. coli andS. agalactiae), wherein E. coli and S. agalactiae together have adecreased odds of resistance to cefepime.

The present invention also features a method of treating a polymicrobialinfection comprising E. coli and S. agalactiae (or a method of killingor inhibiting growth of E. coli and S. agalactiae), wherein the methodcomprises introducing ceftazidime to the E. coli and S. agalactiaepolymicrobial infection (e.g., administering ceftazidime to the patienthaving or suspected of having a polymicrobial infection with E. coli andS. agalactiae), wherein E. coli and S. agalactiae together have adecreased odds of resistance to ceftazidime.

The present invention also features a method of treating a polymicrobialinfection comprising E. coli and S. agalactiae (or a method of killingor inhibiting growth of E. coli and S. agalactiae), wherein the methodcomprises introducing ceftriaxone to the E. coli and S. agalactiaepolymicrobial infection (e.g., administering ceftriaxone to the patienthaving or suspected of having a polymicrobial infection with E. coli andS. agalactiae), wherein E. coli and S. agalactiae together have adecreased odds of resistance to ceftriaxone.

The present invention also features a method of treating a polymicrobialinfection comprising E. coli and S. agalactiae (or a method of killingor inhibiting growth of E. coli and S. agalactiae), wherein the methodcomprises introducing ciprofloxacin to the E. coli and S. agalactiaepolymicrobial infection (e.g., administering ciprofloxacin to thepatient having or suspected of having a polymicrobial infection with E.coli and S. agalactiae), wherein E. coli and S. agalactiae together havea decreased odds of resistance to ciprofloxacin.

The present invention also features a method of treating a polymicrobialinfection comprising E. coli and S. agalactiae (or a method of killingor inhibiting growth of E. coli and S. agalactiae), wherein the methodcomprises introducing levofloxacin to the E. coli and S. agalactiaepolymicrobial infection (e.g., administering levofloxacin to the patienthaving or suspected of having a polymicrobial infection with E. coli andS. agalactiae), wherein E. coli and S. agalactiae together have adecreased odds of resistance to levofloxacin.

The present invention also features a method of treating a polymicrobialinfection comprising E. coli and S. agalactiae (or a method of killingor inhibiting growth of E. coli and S. agalactiae), wherein the methodcomprises introducing tetracycline to the E. coli and S. agalactiaepolymicrobial infection (e.g., administering tetracycline to the patienthaving or suspected of having a polymicrobial infection with E. coli andS. agalactiae), wherein E. coli and S. agalactiae together have adecreased odds of resistance to tetracycline.

The present invention also features a method of treating a polymicrobialinfection comprising E. coli and S. agalactiae (or a method of killingor inhibiting growth of E. coli and S. agalactiae), wherein the methodcomprises introducing TMP/sulfamethoxazole to the E. coli and S.agalactiae polymicrobial infection (e.g., administeringTMP/sulfamethoxazole to the patient having or suspected of having apolymicrobial infection with E. coli and S. agalactiae), wherein E. coliand S. agalactiae together have a decreased odds of resistance toTMP/sulfamethoxazole.

Thus, the methods of the present invention also include treating apolymicrobial infection comprising E. coli and S. agalactiae maycomprise introducing (e.g., administering) one or more antimicrobialsselected from: ampicillin, cefepime, ceftazidime, ceftriaxone,ciprofloxacin, levofloxacin, tetracycline, or TMP/sulfamethoxazole.

The present invention also features a method of guiding treatment of apolymicrobial infection comprising E. coli and S. agalactiae. The methodmay comprise detecting the polymicrobial infection with E. coli and S.agalactiae and providing a report showing one or more antibiotics towhich E. coli and S. agalactiae have increased and/or decreasesresistance to. The report may help the physician choose an appropriateantibiotic to administer to the patient.

E. coli and P. mirabilis

The present invention also features a method of treating a polymicrobialinfection comprising E. coli and P. mirabilis (or a method of killing orinhibiting growth of E. coli and P. mirabilis), wherein the methodcomprises introducing cefaclor to the E. coli and P. mirabilispolymicrobial infection (e.g., administering cefaclor to the patienthaving or suspected of having a polymicrobial infection with E. coli andP. mirabilis), wherein E. coli and P. mirabilis together have adecreased odds of resistance to cefaclor.

The present invention also features a method of treating a polymicrobialinfection comprising E. coli and P. mirabilis (or a method of killing orinhibiting growth of E. coli and P. mirabilis), wherein the methodcomprises introducing cefazolin to the E. coli and P. mirabilispolymicrobial infection (e.g., administering cefazolin to the patienthaving or suspected of having a polymicrobial infection with E. coli andP. mirabilis), wherein E. coli and P. mirabilis together have adecreased odds of resistance to cefazolin.

The present invention also features a method of treating a polymicrobialinfection comprising E. coli and P. mirabilis (or a method of killing orinhibiting growth of E. coli and P. mirabilis), wherein the methodcomprises introducing cefoxitin to the E. coli and P. mirabilispolymicrobial infection (e.g., administering cefoxitin to the patienthaving or suspected of having a polymicrobial infection with E. coli andP. mirabilis), wherein E. coli and P. mirabilis together have adecreased odds of resistance to cefoxitin.

The present invention also features a method of treating a polymicrobialinfection comprising E. coli and P. mirabilis (or a method of killing orinhibiting growth of E. coli and P. mirabilis), wherein the methodcomprises introducing ceftazidime to the E. coli and P. mirabilispolymicrobial infection (e.g., administering ceftazidime to the patienthaving or suspected of having a polymicrobial infection with E. coli andP. mirabilis), wherein E. coli and P. mirabilis together have adecreased odds of resistance to ceftazidime.

The present invention also features a method of treating a polymicrobialinfection comprising E. coli and P. mirabilis (or a method of killing orinhibiting growth of E. coli and P. mirabilis), wherein the methodcomprises introducing ceftriaxone to the E. coli and P. mirabilispolymicrobial infection (e.g., administering ceftriaxone to the patienthaving or suspected of having a polymicrobial infection with E. coli andP. mirabilis), wherein E. coli and P. mirabilis together have adecreased odds of resistance to ceftriaxone.

Thus, the methods of the present invention also include treating apolymicrobial infection comprising E. coli and P. mirabilis may compriseintroducing (e.g., administering) one or more antimicrobials selectedfrom: cefaclor, cefazolin, cefoxitin, ceftazidime, or ceftriaxone.

The present invention also features a method of guiding treatment of apolymicrobial infection comprising E. coli and P. mirabilis. The methodmay comprise detecting the polymicrobial infection with E. coli and P.mirabilis and providing a report showing one or more antibiotics towhich E. coli and P. mirabilis have increased and/or decreasesresistance to. The report may help the physician choose an appropriateantibiotic to administer to the patient.

E. coli and K. pneumoniae

The present invention also features a method of treating a polymicrobialinfection comprising E. coli and K. pneumoniae (or a method of killingor inhibiting growth of E. coli and K. pneumoniae), wherein the methodcomprises introducing levofloxacin to the E. coli and K. pneumoniaepolymicrobial infection (e.g., administering levofloxacin to the patienthaving or suspected of having a polymicrobial infection with E. coli andK. pneumoniae), wherein E. coli and K. pneumoniae together have adecreased odds of resistance to levofloxacin.

The present invention also features a method of treating a polymicrobialinfection comprising E. coli and K. pneumoniae (or a method of killingor inhibiting growth of E. coli and K. pneumoniae), wherein the methodcomprises introducing tetracycline to the E. coli and K. pneumoniaepolymicrobial infection (e.g., administering tetracycline to the patienthaving or suspected of having a polymicrobial infection with E. coli andK. pneumoniae), wherein E. coli and K. pneumoniae together have adecreased odds of resistance to tetracycline.

Thus, the methods of the present invention also include treating apolymicrobial infection comprising E. coli and K. pneumoniae maycomprise introducing (e.g., administering) one or more antimicrobialsselected from: levofloxacin or tetracycline.

The present invention also features a method of guiding treatment of apolymicrobial infection comprising E. coli and K. pneumoniae. The methodmay comprise detecting the polymicrobial infection with E. coli and K.pneumoniae and providing a report showing one or more antibiotics towhich E. coli and K. pneumoniae have increased and/or decreasesresistance to. The report may help the physician choose an appropriateantibiotic to administer to the patient.

E. coli and K. oxytoca

The present invention also features a method of treating a polymicrobialinfection comprising E. coli and K. oxytoca (or a method of killing orinhibiting growth of E. coli and K. oxytoca), wherein the methodcomprises introducing cefepime to the E. coli and K. oxytocapolymicrobial infection (e.g., administering cefepime to the patienthaving or suspected of having a polymicrobial infection with E. coli andK. oxytoca), wherein E. coli and K. oxytoca together have a decreasedodds of resistance to cefepime.

The present invention also features a method of treating a polymicrobialinfection comprising E. coli and K. oxytoca (or a method of killing orinhibiting growth of E. coli and K. oxytoca), wherein the methodcomprises introducing ciprofloxacin to the E. coli and K. oxytocapolymicrobial infection (e.g., administering ciprofloxacin to thepatient having or suspected of having a polymicrobial infection with E.coli and K. oxytoca), wherein E. coli and K. oxytoca together have adecreased odds of resistance to ciprofloxacin.

The present invention also features a method of treating a polymicrobialinfection comprising E. coli and K. oxytoca (or a method of killing orinhibiting growth of E. coli and K. oxytoca), wherein the methodcomprises introducing tetracycline to the E. coli and K. oxytocapolymicrobial infection (e.g., administering tetracycline to the patienthaving or suspected of having a polymicrobial infection with E. coli andK. oxytoca), wherein E. coli and K. oxytoca together have a decreasedodds of resistance to tetracycline.

Thus, the methods of the present invention also include treating apolymicrobial infection comprising E. coli and K. oxytoca may compriseintroducing (e.g., administering) one or more antimicrobials selectedfrom: cefepime, ciprofloxacin, or tetracycline.

The present invention also features a method of guiding treatment of apolymicrobial infection comprising E. coli and K. oxytoca. The methodmay comprise detecting the polymicrobial infection with E. coli and K.oxytoca and providing a report showing one or more antibiotics to whichE. coli and K. oxytoca have increased and/or decreases resistance to.The report may help the physician choose an appropriate antibiotic toadminister to the patient.

The present invention also features methods for treating a patienthaving or suspected of having a polymicrobial infection comprising acombination of E. coli and K. Pneumoniae. In certain embodiments, themethod comprising: detecting the presence of both E. coli and K.Pneumoniae in a source of the infection obtained from the patient; andadministering to the patient an antibiotic other thanampicillin/sulbactam or cefaclor, wherein E. coli and K. Pneumoniaetogether have an increased odds of resistance to ampicillin/sulbactamand cefaclor.

The present invention also features methods for treating a patienthaving or suspected of having a polymicrobial infection comprising acombination of E. faecalis and K. Pneumoniae. In certain embodiments,the method comprises detecting the presence of both E. faecalis and K.Pneumoniae in a source of the infection obtained from the patient; andadministering to the patient an antibiotic other thanamoxicillin/clavulanate or ampicillin/sulbactam, wherein E. faecalis andK. Pneumoniae together have an increased odds of resistance toamoxicillin/clavulanate and ampicillin/sulbactam.

The present invention also features methods for treating a patienthaving or suspected of having a polymicrobial infection comprising acombination of E. faecalis and S. agalactiae. In certain embodiments,the method comprises detecting the presence of both E. faecalis and S.agalactiae in a source of the infection obtained from the patient; andadministering to the patient an antibiotic other than tetracyclinewherein E. faecalis and S. agalactiae together have an increased odds ofresistance to tetracycline.

The present invention also features methods for treating a patienthaving or suspected of having a polymicrobial infection comprising acombination of E. coli and CoNS. In certain embodiments, the methodcomprises detecting the presence of both E. coli and CoNS in a source ofthe infection obtained from the patient; and administering to thepatient an antibiotic other than levofloxacin, wherein E. coli and CoNStogether have an increased odds of resistance to levofloxacin.

The present invention is not limited to the aforementioned polymicrobialinfections and administered antibiotics.

Any feature or combination of features described herein are includedwithin the scope of the present invention provided that the featuresincluded in any such combination are not mutually inconsistent as willbe apparent from the context, this specification, and the knowledge ofone of ordinary skill in the art. Additional advantages and aspects ofthe present invention are apparent in the following detailed descriptionand claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The patent application or application file contains at least one drawingexecuted in color. Copies of this patent or patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee.

The features and advantages of the present invention will becomeapparent from a consideration of the following detailed descriptionpresented in connection with the accompanying drawings in which:

FIG. 1 shows the effects of E. coli and K. pneumoniae interactions onresistance to ampicillin/sulbactam, cefaclor, and tetracycline.

FIG. 2 shows the effects of organism interactions on antibioticresistance. An upward arrow indicates an increase in resistance. Adownward arrow indicates a decrease in resistance (an increase insusceptibility).

FIG. 3 depicts examples of polymicrobial infections that experiencedecreases or increases in antibiotic sensitivity, relative tomonomicrobial infections.

FIG. 4 is a continuation of FIG. 3, depicting examples of polymicrobialinfections that experience decreases or increases in antibioticsensitivity, relative to monomicrobial infections.

FIG. 5 depicts various odds ratios of resistance, comparingpolymicrobial infections with monomicrobial infections.

FIG. 6 depicts examples of polymicrobial infections that experiencedecreases or increases in antibiotic sensitivity, relative tomonomicrobial infections.

FIG. 7 is a continuation of FIG. 6, depicting examples of polymicrobialinfections that experience decreases or increases in antibioticsensitivity, relative to monomicrobial infections.

FIG. 8 is a continuation of FIG. 6 and FIG. 7, depicting examples ofpolymicrobial infections that experience decreases or increases inantibiotic sensitivity, relative to monomicrobial infections.

FIG. 9 shows correlations between organisms found in polymicrobialinfections, particularly polymicrobial infections with 2, 3, 4, 5, 6, or7 organisms.

FIG. 10 shows correlations between organisms found in polymicrobialinfections, excluding E. coli. The strength of correlation isrepresented by the width of the edge connecting the genes. Onlycorrelations greater than 0.1 are shown.

FIG. 11 depicts an exemplary Antibiotic Source Plate with well contentsand antibiotic concentration (μg/mL). Nitro=nitrofurantoin,Cipro=ciprofloxacin, Mero=meropenem, Ceftiaxone=ceftriaxone,TMP/SMX=trimethoprim+sulfamethoxazole, Pip/Tazo=piperacillin+tazobactam,Levo=levofloxacin, Cefoxitin=cefoxitin, Tetra=tetracycline,Amp/Sulb=ampicillin+sulbactam, Amp=ampicillin, and Vanco=vancomycin.

FIG. 12 depicts an exemplary Antibiotic Source Plate with well contentsand antibiotic concentration (μg/mL). Cefazolin=cefazolin,Cefepime=cefepime, Ceftazidime=ceftazidime, Gentamicin=gentamicin,Amox/Clav=amoxicillin+clavulanate, Cefaclor=cefaclor.

TERMS

As used herein, the term “Highest Single Agent Interaction Principle”refers to a statistical model wherein the resistance of thepolymicrobial infection is predicted to be the resistance of thebacteria with the highest resistance. For example, if species A isresistant with a probability 20%, and species B is resistant with aprobability 50%, then the probability of resistance of the pool is 50%.

As used herein, the term “Union Principle” refers to a statistical modelwherein the polymicrobial infection of species A and B is made up of onecolony (or one genetic variant) of species A and one colony (or onegenetic variant) of species B, and the polymicrobial infection isresistant if either the colony of species A is resistant, or if thecolony of species B is resistant. For example, if an antibiotic isapplied to the polymicrobial infection, it may kill off species A, butif species B survives, the polymicrobial infection is called resistant.For example, if species A is resistant with a probability 20%, andspecies B is resistant with a probability 50%, then the probability ofresistance of the pool is:

P(pool resistance)=P(A)+P(B)−P(A and B)

As used herein, the term “Logistic Additive Model” refers to astatistical model wherein the effects of species A and species B on theresistance of the polymicrobial infection is estimated in a logisticmodel. The effect of species A is the odds ratio of resistance whenspecies A is present relative to when it is not present; similarly, theeffect of species B is the odds ratio of resistance when species B ispresent relative to when it is not. The additive model predicts theeffect of both species as the sum of the log odds-ratio; or the productof the two individual odds-ratios. For example, if the backgroundresistance rate is 50%, the expected polymicrobial infection (species Aand B) resistance with no interactions is 20%; if the backgroundresistance rate is 20%, the expected polymicrobial infection resistanceis 50%.

DETAILED DESCRIPTION OF THE INVENTION Antibiotics

The present methods are conducted using a plurality of antibioticsselected from the large number available to treat patients. Antibiotics(also referred to as anti-microbial agents or anti-bacterial agents)include, but are not limited to, penicillins, tetracyclines,cephalosporins, quninolones, lincomycins, macrolides, sulronamides,glycopeptide antibiotics, aminoglycosides, carbapenems, ansamycins,lipopeptides, monobactams, nitrofurans, oxaxolidinones, andpolypeptides.

Penicillin antibiotics include, but are not limited to, penicillin,methicillin, amoxicillin, ampicillin, flucloxacillin, penicillin G,penicillin V, carbenicillin, piperacillin, ticarcillin, oxacillin,dicloxacillin, azlocillin, cloxacillin, mezlocillin, temocillin, andnafcillin. Additionally, penicillin antibiotics are often used incombination with beta-lactamase inhibitors to provide broader spectrumactivity; these combination antibiotics include amoxicillin/clavulanate,ampicillin/sulbactam, piperacillin/tazobactam, andclavulanate/ticarcillin.

Tetracycline antibiotics include, but are not limited to, tetracycline,doxycycline, demeclocycline, minocycline, and oxytetracycline.

Cephalosporin antibiotics include, but are not limited to, cefadroxil,cephradine, cefazolin, cephalexin, cefepime, ceftaroline, loracarbef,cefotetan, cefuroxime, cefprozil, cefoxitin, cefaclor, ceftibuten,cetriaxone, cefotaxime, cefpodoxime, cefdinir, cefixime, cefditoren,ceftizoxime, cefoperazone, cefalotin, cefamanadole, ceftaroline fosamil,cetobiprole, and ceftazidime. Cephalosporin antibiotics are often usedin combination with beta-lactamase inhibitors to provide broaderspectrum activity; these combination antibiotics include, but are notlimited to, avibactam/ceftazidime and ceftolozane/tazobactam.

Quinolone antibiotics include, but are not limited to, lomefloxacin,ofloxacin, norfloxacin, gatifloxacin, ciprofloxacin, moxifloxacin,levofloxacin, gemifloxacin, cinoxacin, nalidixic acid, trovaloxacin,enoxacin, grepafloxacin, temafloxacin, and sparfloxacin.

Lincomycin antibiotics include, but are not limited to, clindamycin andlincomycin.

Macrolide antibiotics include, but are not limited to, azithromycin,clarithromycin, erythromycin, telithromycin, dirithromycin,roxithromycin, troleandomycin, spiramycin, and fidazomycin.

Sulfonamide antibiotics include, but are not limited to,sulfamethoxazole, sulfasalazine, mafenide, sulfacetamide, sulfadiazine,silver sufadiazine, sulfadimethoxine, sulfanilimide, sulfisoxazole,sulfonamidochrysoidine, and sulfisoxazole. Sulfonamide antibiotics areoften used in combination with trimethoprim to improve bactericidalactivity.

Glycopeptide antibiotics include, but are not limited to, dalbavancin,oritavancin, telavancin, teicoplanin, and vancomycin.

Aminoglycoside antibiotics include, but are not limited to, paromomycin,tobramycin, gentamicin, amikacin, kanamycin, neomycin, netilmicin,streptomycin, and spectinomycin.

Carbapenem antibiotics include, but are not limited to, imipenem,meropenem, doripenem, ertapenem, and imipenem/cilastatin.

Ansamycin antibiotics include, but are not limited to, geldanamycin,herbimycin, and rifaximin.

Lipopeptide antibiotics include, but are not limited to, daptomycin.

Monobactam antibiotics include, but are not limited to, aztreonam.

Nitrofuran antibiotics include, but are not limited to furazolidone andnitrofurantoin.

Oxaxolidinone antibiotics include, but are not limited to, linezolid,posizolid, radezolid, and torezolid.

Polypeptide antibiotics include, but are not limited to, bacitracin,colistin, and polymyxin B.

Other antibiotics which are not part of any of the above-mentionedgroups include, but are not limited to, clofazimine, dapsone,capreomycin, cycloserine, ethambutol, ethionamide, isoniazid,pyrazinamide, rifampicin, rifabutin, rifapentine, streptomycin,arsphenamide, chloramphenicol, fosfomycin, fusidic acid, metronidazole,mupirocin, platensimycin, quinupristin/dalfopristin, thiamphenicol,tigecycline, tinidazole, and trimethoprim.

Additionally, the scope of the presently disclosed methods encompassesthe inclusion of antibiotics not yet known, or not yet approved byregulatory authorities. The presently claimed assay can be performedwith any anti-bacterial agent and is not limited to the antibioticsdisclosed herein.

Treatment of Polymicrobial Infections

Disclosed herein are methods for detecting polymicrobial infections aswell as treating polymicrobial infections, wherein a mixed population ofmicrobes (e.g., bacteria) are present in a patient sample and themicrobes are not first isolated from the sample.

For example, the present invention describes specific polymicrobialinfections and methods of treating said infections, wherein a particularantibiotic or a group of antibiotics are selected based on thecomposition of the polymicrobial infections. As is described below,certain polymicrobial infections show a surprising reduction inantibiotic resistance relative to what might be expected. For example,Klebsiella has a relatively high resistance rate to ampicillin. However,it was surprisingly found that Klebsiella combined withcoagulase-negative Staphylococcus (CNS or CoNS) have a reducedresistance to ampicillin relative to what might be expected (e.g., basedon the union principle or single highest agent principle).

FIG. 1 shows a detailed example of the combinations of E. coli and K.pneumoniae and the effects on resistance to ampicillin/sulbactam,cefaclor, and tetracycline. When combined, E. coli and K. pneumoniaehave a higher resistance to ampicillin/sulbactam and cefaclor relativeto what would be expected (e.g., based on the union principle or singlehighest agent principle). However, E. coli and K. pneumoniae togetherhave a reduced resistance to tetracycline relative to what would beexpected (e.g., based on the union principle or single highest agentprinciple).

FIG. 2 shows the effects of organism interactions on antibioticresistance for a variety of microbes and antibiotics. An upward arrowindicates an increase in resistance (e.g., a decrease insusceptibility). A downward arrow indicates a decrease in resistance(e.g., an increase in susceptibility). Examples of organism interactionsthat result in a decrease in resistance (e.g., increase in susceptibly)includes but is not limited to:

Coagulase-negative Staphylococcus (CONS) and E. coli, orcoagulase-negative Staphylococcus (CONS) and E. faecalis, orcoagulase-negative Staphylococcus (CONS) and K. pneumoniae, or E.faecalis and E. coli show a decrease in resistance toamoxicilliniclavulanate. E. coli and S. agalactiae, or E. faecalis andS. agalactiae show a decrease in resistance to ampicillin. E. faecalisand E. coli show a decrease in resistance to ampicillin/sulbactam. E.coli and P. mirabilis show a decrease in resistance to cefaclor orcefazolin. E. coli and K. oxytoca, or E. coli and S. agalactiae show adecrease in resistance to cefepime. E. coli and P. mirabilis show adecrease in resistance to cefoxitin. E. coli and P. mirabilis, or E.coli and S. agalactiae show a decrease in resistance to ceftazidime.

Coagulase-negative Staphylococcus (CoNS) and E. coli, orCoagulase-negative Staphylococcus (CONS) and K. pneumoniae, orCoagulase-negative Staphylococcus (CoNS) and S. agalactiae, or E. coliand P. mirabilis, or E. coli and S. agalactiae show a decrease inresistance to ceftriaxone.

Coagulase-negative Staphylococcus (CONS) and K. pneumoniae, orCoagulase-negative Staphylococcus (CoNS) and S. agalactiae, or E. coliand K. oxytoca, or E. coli and S. agalactiae show a decrease inresistance to ciprofloxacin.

Coagulase-negative Staphylococcus (CoNS) and E. coli, orCoagulase-negative Staphylococcus (CoNS) and K. pneumoniae, orCoagulase-negative Staphylococcus (CoNS) and S. agalactiae, or E. coliand S. agalactiae, or E. faecalis and K. pneumoniae show a decrease inresistance to levofloxacin.

Coagulase-negative Staphylococcus (CoNS) and E. faecalis, orCoagulase-negative Staphylococcus (CoNS) and K. pneumoniae, orCoagulase-negative Staphylococcus (CoNS) and S. agalactiae show adecrease in resistance to gentamicin.

E. faecalis and E. coli, or E. faecalis and K. pneumoniae, or E.faecalis and P. aeruginosa, or E. faecalis and P. mirabilis show adecrease in resistance to meropenem. Coagulase-negative Staphylococcus(CoNS) and E. coli, or Coagulase-negative Staphylococcus (CoNS) and E.faecalis, or Coagulase-negative Staphylococcus (CoNS) and S. agalactiae,or E. coli and K. oxytoca, or E. coli and K. pneumoniae, or E. coli andS. agalactiae, or E. faecalis and E. coli, or E. faecalis and K.pneumoniae show a decrease in resistance to tetracycline.Coagulase-negative Staphylococcus (CoNS) and E. coli, orCoagulase-negative Staphylococcus (CoNS) and K. pneumoniae, orCoagulase-negative Staphylococcus (CONS) and S. agalactiae, or E.faecalis and E. coli show a decrease in resistance toTMP/sulfamethoxazole. Coagulase-negative Staphylococcus (CONS) and E.faecalis, or Coagulase-negative Staphylococcus (CONS) and S. agalactiae,or E. faecalis and S. agalactiae show a decrease in resistance tovancomycin.

FIG. 3, FIG. 4, FIG. 5, FIG. 6, FIG. 7, and FIG. 8 also show effects oforganism interactions on antibiotic resistance. FIG. 9 and FIG. 10 showcorrelations between the presence of particular organisms found inpolymicrobial infections.

Thus, the present invention describes methods of treating polymicrobialinfections, such as the aforementioned polymicrobial infections, withappropriate antibiotics, such as the antibiotics to which thepolymicrobial infections have decreased resistance. The methods hereinmay comprise the detection of the presence of the combination of thebacteria (e.g., the two or more bacteria in a polymicrobial infection)in a source of the infection obtained from the patient. The detectionprocess may not necessarily involve first isolating each bacterium fromthe source of infection.

For example, the present invention features methods of treating apatient having or suspected of having a polymicrobial infectioncomprising a combination of E. faecalis and E. coli. In certainembodiments, the method comprises administering one or a combination ofamoxicillin/clavulanate, ampicillin/sulbactam, levofloxacin, meropenem,or tetracycline.

The present invention also features methods for treating a patienthaving or suspected of having a polymicrobial infection comprising acombination of E. faecalis and K. pneumoniae. In certain embodiments,the method comprises administering one or a combination of meropenem ortetracycline.

The present invention also features methods for treating a patienthaving or suspected of having a polymicrobial infection comprising acombination of E. faecalis and P. aeruginosa. In certain embodiments,the method comprises administering meropenem to the patient.

The present invention also features methods for treating a patienthaving or suspected of having a polymicrobial infection comprising acombination of E. faecalis and P. miribilis. In certain embodiments, themethod comprises administering meropenem to the patient.

The present invention also features methods for treating a patienthaving or suspected of having a polymicrobial infection comprising acombination of E. faecalis and S. agalactiae. In certain embodiments,the method comprises administering one or a combination of ampicillin orvancomycin.

The present invention is not limited to the aforementioned examples andencompasses any example described herein, including the figures.

Antibiotic Resistance (ABR) Testing Plates

Referring to other disclosed methods herein, samples may be collectedfrom subjects according to standard collection protocols in sterilecontainers and are transported to the testing facility.

An example of the preparation of the antibiotic resistance (ABR) testingplates involves two steps. First is preparation of antibiotic solutionsand the second is preparation of the bacterial growth medium plate. Theantibiotics to be tested for any given sample include antibiotics knownto be useful for treating the tissue having the suspected infection, orany antibiotics requested by a medical or laboratory professional havingknowledge of the particular patient sample. It is anticipated that mostassays will be performed with a standard panel of antibiotics based onthe type and location of infection suspected by a medical professional.In some embodiments, the standard panel of antibiotics comprisesnitrofurantoin, ciprofloxacin, meropenem, ceftriaxone,trimethoprim/sulfamethoxazole, piperacillin/tazobactam, levofloxacin,cefoxitin, tetracycline, ampicillin/sulbactam, ampicillin, andvancomycin. However, patients with known antibiotic allergies orsensitivities, or with a history of antibiotic resistance, may requirecustomized panels of antibiotics. The assay can be performedsimultaneous with an unlimited number of antibiotics.

Antibiotic stock solutions are prepared using solvents suitable for eachantibiotic and then 10× solutions are prepared and stored in multi-wellplates to allow efficient transfer to testing plates. Each antibiotic istested at a minimum of concentrations. In some embodiments, threeconcentrations, four concentrations, five concentrations, sixconcentrations, seven concentrations, eight concentrations, nineconcentrations, or ten concentrations of an antibiotic, or antibioticcombination, are included in the assay. Typically serial dilutions ofthe antibiotics are prepared wherein each dilution represents half theconcentration of the higher concentration. The 10× antibiotic solutionsare stored in the multi-well plate according to a plate plan establishedfor the antibiotic panel chosen for the assay. Exemplary plate plans aredepicted in the Antibiotic Source Plates in FIG. 11 and FIG. 12.Antibiotic stocks and 10× solutions are stored at 2-8° C. until needed.

The ABR testing plates may be multi-well plates (e.g., 6-well, 12-well,24-well, 48-well, 96-well, 384-well plates, or any multi-well platesuitable for this purpose) capable of containing bacterial growth mediumand culturing bacteria. In some embodiments, the plates are 96-wellplates. In some embodiments, sterile agar-bacterial growth medium isdispensed into each well of the plate. Exemplary agar-bacterial growthmedium include, but are not limited to Mueller-Hinton agar, blood agar,trypticase soy agar, etc. After the agar has solidified at roomtemperature, 1/10 volume (of bacterial growth medium) of 10× antibioticsolution is added to each well of the test plate according to thepre-determined plate plan. After the antibiotics have been introduced tothe bacterial growth medium, the plates are allowed to rest for at leastone hour. For long-term storage, the antibiotic-containing ABR platesare stored at 2-8° C. In some embodiments, sterile liquid brothbacterial growth medium mixed with sample is dispensed into each well ofthe plate containing 1/10 volume (of bacterial growth medium) of 10×antibiotic solution arrayed according to a pre-determined plate plan.Multi-well plates containing 1/10 volume (of final well volume ofbacterial growth medium and antibiotic solution) are stored at 2-8° C.for later use or long-term storage.

Samples for the disclosed antibiotic resistance testing may beoptionally diluted in sterile aqueous solution or mixed with bacterialgrowth medium. In some embodiments, a volume of sample for the disclosedantibiotic resistance testing are first mixed with a growth medium andincubated for 0-24 hours at an incubation temperature of 35±4° C. Thesamples are then diluted with saline and then mixed with growth mediumand added to room temperature ABR testing plates at 9/10 volume of eachwell in the multi-well plate. In some embodiments, samples are added toroom temperature ABR plates at 1/20 volume of bacterial growth mediumpresent in the well. A single patient specimen is used for each ABRplate. If multiple patient specimens are to be tested, each specimen isassayed in its own plate. Once inoculated, the plates are covered andincubated to encourage bacterial growth. Embodiments where a singlesample is assayed using more than one plate are also within the scope ofthe present method.

The plates can be used to culture either anaerobic or aerobic bacteria.For culture of anaerobic bacteria, the plates are incubated at atemperature and in a reduced-oxygen environment to encourage growth ofanaerobic bacteria. For culture of aerobic bacteria, the plates areincubated at a temperature and in an oxygen-containing environment toencourage growth of aerobic bacteria.

The incubation temperature can vary depending on the expected types ofbacteria but will most likely be in a range of 35-40° C. The platescontaining samples are incubated for 12-48 hours, 12-24 hours, 24-28hours, 12-36 hours, 14-30 hours, 16-24 hours, 16-20 hours, or 16-18hours, or any range bounded by these numbers.

In some embodiments, wherein the assay is performed with anagar-containing medium, after incubation, bacteria present in each wellare recovered by resuspension in an aqueous liquid. Suitable liquidsinclude, but are not limited to, water, saline, culture medium, etc. Theaqueous liquid should be sterile, or at least free from bacterialgrowth. A volume of liquid equal to 100% of the volume of bacterialgrowth medium is carefully added to the wells of the ABR plate andallowed to sit for at least 30 minutes. In some embodiments, the platesare allowed to sit for 35 minutes, 40 minutes, 45 minutes, 50 minutes,or 60 minutes. The resulting suspension is then carefully removed fromeach well into individual wells of a clean multi-well plate according tothe predetermined plate plan. The plates are optionally agitated tocause mixing of the bacteria with the liquid prior to removal of thesuspension. In some embodiments wherein the assay is performed usingliquid growth medium, the multi-well plate will be applied to OD₆₀₀measurement immediately after incubation.

The multi-well plate containing the bacteria-containing suspension isthen read in a spectrophotometer. The optical density of the recoveredliquid is measured at OD₆₀₀ multiple times to correct for unevendistribution of bacteria particles in the suspension. In someembodiments, the plates are read one time, two times, three times, fourtimes, five times, six times, seven times, or eight times. The multipleplate reads occur in sequence without allowing the suspension to settlein the wells.

The multiple OD₆₀₀ of each well are averaged to provide an accuratequantitation of bacteria present in each well under the specificconditions. Each well's average OD₆₀₀ is then adjusted for background bysubtracting the average OD₆₀₀ measurements of a well where no bacteriacould grow to yield a blanked value. In some embodiments, this no-growthwell contains a blend of antibiotics (AB-blend). In some embodiments,this no-growth well contains sodium azide (Na-Azide). The blanked valueis representative of the ability of bacteria to grow in the presence ofthe particular antibiotic in the well.

The blanked results are then converted into a “resistance” (R) or“sensitive” (S) score based on a threshold value. OD₆₀₀ measurementsgreater than or equal to the threshold are interpreted as resistant,while measurements below the threshold are interpreted as sensitive.

In some embodiments, the threshold value is for an agar-containingmedium. In some embodiments, a threshold value has been determined at0.010 to 1.000, 0.010-0.090, 0.015 to 0.035, or 0.020 to 0.030 based oncorrelations to a standard reference method. In some embodiments, thethreshold value as been determined at about 0.010, about 0.015, about0.020, about 0.025, about 0.030, about 0.035, about 0.040, about 0.045,about 0.050, about 0.055, about 0.060, about 0.065, about 0.070, about0.075, about 0.080, about 0.085, or about 0.090 based on correlations toa standard reference method. In some embodiments, a threshold value hasbeen determined at 0.025 based on correlations to a standard referencemethod.

In some embodiments, the threshold value is for a liquid medium. In someembodiments, a threshold value has been determined at 0.010-1.000,0.020-0.090, 0.050-0.080, 0.055 to 0.075, or 0.060 to 0.070 based oncorrelation to a consensus score between two standard reference methods.In some embodiments, the threshold value as been determined at about0.010, about 0.015, about 0.020, about 0.025, about 0.030, about 0.035,about 0.040, about 0.045, about 0.050, about 0.055, about 0.060, about0.065, about 0.070, about 0.075, about 0.080, about 0.085, about 0.090,or about 0.095 based on correlation to a consensus score between twostandard reference methods. In some embodiments, a threshold value hasbeen determined at 0.065 based on correlation to a consensus scorebetween two standard reference methods.

In other embodiments, any adjusted OD₆₀₀ measurement greater than blankOD₆₀₀ measurement can be determined as indicative of bacterial growthand applied as a threshold value by correlation to a standard referencemethod or combination of reference methods.

Minimal inhibitory concentrations for each effective antibiotic are thencalculated based on the sensitivity or resistance of the culture at themultiple antibiotic concentrations.

Results of the antibiotic resistance assay disclosed herein aretransmitted to the appropriate medical professional who then has theoption of prescribing an antibiotic, or antibiotics, shown to be activeagainst the patient's infection, changing the antibiotic to a moreeffective antibiotic, or ordering additional testing.

EXAMPLES

The following are non-limiting examples of the present invention. It isto be understood that said examples are not intended to limit thepresent invention in any way. Equivalents or substitutes are within thescope of the present invention.

Example 1. Bacterial Organism Interactions as Detected by PooledAntibiotic Susceptibility Testing (P-AST) in Polymicrobial UrineSpecimens

The standard of care for the diagnosis of UTI is a standard urineculture and sensitivity testing (SUC), and has served to guide treatmentsince the early 1950's. The methodology relies on an “Escherichia coli(E. coli) centric” view of infections that suggested that UTI's arecaused by a single or perhaps two pathogens. Recent findings haveunderscored not only are the vast majority of uropathogens missed byroutine culture but that up to 39% of these infections are polymicrobialin nature. In conjunction, antibiotic resistance has been well-studiedin monomicrobial infections, but has been less well characterized inpolymicrobial infections in the clinical setting. Yet, interactionsbetween organisms can alter responses to antibiotics.

Currently, antibiotic susceptibility testing (AST) ignores bacterialinteractions. In AST, each bacterium is tested in isolation against anantibiotic, providing no opportunity to assess bacterial interactions.Ignoring bacterial interactions can either lead to potential treatmentfailure or prevent the use of efficacious antibiotics. Both scenarioscan have serious clinical consequences. Pooled Antibiotic SusceptiblyTesting (P-AST), on the other hand, permits the ability to considerinteractions since the assay involves simultaneously growing thedetected organisms together in the presence of antibiotics and thenmeasures susceptibility. P-AST may therefore provide therapeutic optionsthat take into consideration the ability of organisms to respond toantibiotics in conjunction with bacterial interactions.

The present invention describes Pooled Antibiotic Susceptibility Testing(P-AST), which involves simultaneously growing all detected bacteriatogether in the presence of antibiotics and then measuringsusceptibility. Thus, P-AST considers interactions between cohabitingbacterial species. Urine specimens were obtained from patientspresenting with UTI-like symptoms to 37 urology clinics. The odds ofresistance were estimated for 18 antibiotics relative to increasingnumbers of bacterial species in a specimen. It was found thatantimicrobial susceptibility patterns in polymicrobial specimensdiffered from those observed in monomicrobial specimens. Since standardof care relies on assessment of antibiotic susceptibility inmonomicrobial infections, these findings show that P-AST could serve asa more accurate predictor of antibiotic susceptibility.

The following study combines data from two studies of antibioticresistance patterns in elderly patients presenting with symptomsconsistent with a UTI. Retrospective data and patient information wereobtained from a single site for 613 patients. Prospective data andpatient information were obtained for 2,511 patients who presented atany of 37 geographically disparate clinics in the United States. Allsubjects met the following inclusion and exclusion criteria. Inclusioncriteria included: symptoms of acute cystitis, complicated UTI,persistent UTI, recurrent UTI, prostatitis, pyelonephritis, interstitialcystitis (at any age), symptoms of other conditions at years of age,specimen volumes sufficient to permit urine culture and MultiplexPolymerase Chain Reaction (M-PCR) combined with Pooled AntibioticSensitivity Testing (P-AST), patient informed consented, documentedtimes at which the specimens were collected and stabilized with boricacid in grey-top tubes. Exclusion criteria included prior participationin this study, antibiotics taken for any reason other than UTI at thetime of enrollment, chronic (≥10 days) indwelling catheters,self-catheterization, and urinary diversion. Antibiotic susceptibilitydata were available for 1,352 of the 3,124 patients (43.3%).

DNA extraction was performed using the KingFisher/MagMAX™ Automated DNAExtraction instrument and the MagMAX™ DNA Multi-Sample Ultra Kit(ThermoFisher, Carlsbad, Calif.). 1 mL of urine were transferred to96-well deep-well plates, sealed, and centrifuged to concentrate thesamples, and then the supernatant was removed. Enzyme Lysis Mix (200μL/well) was added to the samples, which were then incubated for 20 minat 65° C. Proteinase K Mix (PK Mix) was added (50 μL/well) and incubatedfor 30 min at 65° C. Lysis buffer (125 μL/well) and DNA Binding Bead Mix(40 μL/well) were added, and the samples were vortexed for a minimum of5 min. Each 96-well plate was loaded into the KingFisher/MagMAXAutomated DNA Extraction instrument, which was operated in accordancewith standard operating procedures.

DNA analysis was conducted using the Guidance® UTI Test (Pathnostics,Irvine, Calif.), which consists of both M-PCR and P-AST. Samples weremixed with universal PCR master mix and amplified using TaqMantechnology on the Life Technologies 12K Flex OpenArray System™ (LifeTechnologies, Carlsbad, Calif.). DNA samples were spotted in duplicateon 112-format OpenArray chips. Plasmids unique to each bacterial speciesbeing tested were used as positive controls. Any appropriate target maybe usable as an inhibition control, e.g., Candida tropicalis, B.atrophaeus, etc. A data analysis tool developed by Pathnostics was usedto sort data, assess the quality of data, summarize control sample data,identify positive assays, calculate concentrations, and generate draftreports. Probes and primers were used to detect the following pathogenicbacteria: Acinetobacter baumannii, Actinotignum schaalii, Aerococcusurinae, Alloscardovia omnicolens, Citrobacter freundii, Citrobacterkoseri, Corynebacterium riegelii, Enterobacter aerogenes, Enterococcusfaecalis, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae,Morganella morganii, Mycobacterium tuberculosis, Mycoplasma genitalium,Mycoplasma hominis, Pantoea agglomerans, Proteus mirabilis, Providenciastuartii, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcusaureus, Streptococcus agalactiae, and Ureaplasma urealyticum. Probes andprimers also were used to detect the following bacterial groups:Coagulase negative staphylococci (CONS) (Staphylococcus epidermidis,Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcussaprophyticus); Viridans group streptococci (VGS) (Streptococcusanginosus, Streptococcus oralis, Streptococcus pasteuranus).

The Pooled Antibiotic Sensitivity Test (P-AST) is permitting theorganisms identified to grow in the presence of antibiotics andmeasuring the minimum concentration of antibiotic to inhibit growth.Antibiotic susceptibility testing is performed when at least a singleorganism within a pool of organisms reaches a certain threshold, (e.g.,at least 3,000 cells/ml, at least 5,000 cells/ml, at least 10,000cells/ml, etc.) and can grow in the presence of the antibiotic in theassay within the time of testing. (Note: 10,000 cells/ml is equivalentto 10,000 CFU/ml.) The present invention is not limited to a 10,000cells/mL threshold. These pools of organism(s) are inoculated intogrowth medium then placed onto an antibiotic laden “spec plate” that isthen incubated and analyzed for growth. The breakpoints are derived fromthe CLSI breakpoints.

P-AST was performed by aliquoting 1 mL of patient urine specimen into a1.7 mL microcentrifuge tube. After centrifugation, the supernatant wasaspirated and discarded, leaving approximately 100 or 50 μL (minimumvolume left above pellet created from centrifugation) of patient samplein the microcentrifuge tube. One mL of Mueller Hinton Growth Media wasthen aliquoted into the patient sample in the microcentrifuge tube andthe tubes were incubated at 35° C. in a non-CO₂ incubator for 6 hours.Non-inoculated liquid MH-media is incubated as the negative control toconfirm the media used is not contaminated. Those samples that reached aminimum threshold of 10,000 cells/mL were then diluted by aliquoting 0.5mL of sample into a 50 mL conical tube containing Mueller Hinton GrowthMedia (in this example, the dilution was 1:60, e.g., 500 μL was added to29.5 mL). 96-well plates pre-loaded with antibiotics were theninoculated with diluted samples and incubated along with control platesfor 12-16 hours at 35° C. in a single layer. Optical density of sampleswas then read on a DensiCHEK plate reader. The DensiCHEK instrument isused to measure the concentration of turbid samples considered positivebecause PCR-ID result was >=10,000 cells/mL. The turbid samples werediluted/normalized to the same concentration and then added to a volumeof liquid, e.g., 29.5 mL of liquid MH-media. A spectrophotometer (platereader) is used to measure optical density of the samples in wells ofantibiotics and inoculum in order to determine if there is growth in awell. The present invention is not limited to a concentration of 10,000cells/mL.

Logistic regression was used to compare resistance rates inmonomicrobial and polymicrobial infections. Specifically, 18 differentlogistic regression models were fit to the data: the response variablewas an indicator of whether the specimen was resistant to the specificantibiotic or not and the predictor variable was an indicator of whetherthe infection was monomicrobial or polymicrobial. Specimens wereclassified as monomicrobial if a single bacterial species was detectedabove the 10000 cells/mL threshold; they were classified aspolymicrobial if two or more distinct bacteria species were detectedabove that threshold. Similar logistic regression models also were run,using the number of distinct bacterial species as the predictorvariable. The present invention is not limited to a concentration of10,000 cells/mL.

Interactions between pairs of bacterial species were investigated usinga logistic regression model to predict resistance in the presence ofspecific bacterial species. Bacterial species that tested positive in atleast 30 samples were considered in the regression model: these 16species were A. schaalii, A. urinae, A. omnicolens, CoNS, C. riegelii,E. faecalis, E. coli, K. oxytoca, K. pneumoniae, M. morganii, P.mirabilis, P. aeruginosa, S. aureus, S. agalactiae, U. urealyticum, VGS.Backward stepwise model selection was performed on the model with allmain effects and all pairwise interactions using an enter significancelevel of α=0.10 and an exit significance level of α=0.05 to obtain thebest fitting model. This model was used to predict resistance rates whena specific organism was present or when a specific pair of organismswere present.

Using the logistic regression model described above, the resistance ratefor a pair of species was compared to the resistance rates for eachspecies alone. Two different principles were applied to calculate theexpected resistance rate to a pair of species that do not interact: (a)Highest Single Agent Principle (HSAP) (a commonly used model for druginteractions) and (b) Union Principle (UP) (also used to model druginteractions).

Using the HSAP, a pair of organisms was considered to have aninteraction if the resistance rate of the pair of organisms wasstatistically different from the highest resistance rate of each of thetwo organisms. This principle is based on the idea that a pool of twoorganisms will survive application of a specific antibiotic only if themore resistant bacteria survives. When antibiotic is applied to thepool, it may kill off species A, but if species B survives, the pool iscalled resistant.

The UP assumes a pair of bacteria (species A and B) is made up of onegenetic variant of species A and one genetic variant of species B, andthat the pool is resistant if either species A is resistant or ifspecies B is resistant. If species A is resistant with probability P(A),and species B is resistant with probability P(B), then the probabilityof resistance of the pool is:

P(pool resistance)=P(A)+P(B)−P(A)P(B)

This assumes the two species do not interact, and therefore actindependently. Interactions were statistically tested using bootstrappedsamples of the 3,124 patients: each patient was randomly selected withreplacement. A logistic regression with terms in the best fitting modelselected as above was fit to each bootstrapped sample. The predictedresistance when the pair of organisms was present was compared to thepredicted resistance to each organism alone using the model fit to thebootstrap sample and either the HSAP or UP. 5,000 bootstrapped sampleswere generated and analyzed. If 97.5% or greater of the bootstrappedsamples demonstrated a pool resistance higher than expected, theinteraction was deemed to show a statistically significant interactionwith increased resistance. If 97.5% or greater of the bootstrappedsamples demonstrated a pool resistance lower than expected, theinteraction was deemed to show a statistically significant interactionwith decreased resistance.

A total of 3,124 patients, from two studies and from 37 geographicallydisparate urology clinics in the United States, presenting with symptomsconsistent with a UTI, were initially included in the study. P-AST datawere available for 43.3% (1352) of these patients. Their mean age was 75years. Sixty-six percent (887/1,352) were female, whereas 34%(465/1,352) male.

By M-PCR, 38.9% (1,214/3,124) of the specimens were negative forbacteria, whereas 61.1% (1910/3124) were positive. P-AST data wereavailable for 1,352 (70.7%) of these 1,910 positive specimens. Of these1,352 specimens, 43.9% (594/1352) were monomicrobial, whereas 56.1%(758/1352) were polymicrobial.

Five hundred and fifty eight positive samples lacked antimicrobialsusceptibility data for the following reasons: (1) no species weredetected at ≥10,000 cells/mL (correlating to colony forming units/mL)and thus no species were tested against antibiotics; (2) the speciesdetected by PCR were fastidious (i.e., they required specific growthconditions, extremely restrictive growth conditions, or extreme lengthof time in order to perform susceptibility testing); (3) priorantimicrobial use caused bacteria to fail to thrive in the P-AST assay;or (4) species were not identified because the M-PCR reaction wasinhibited (based upon comparison to negative and positive controls).M-PCR inhibition can occur when an interfering substance prevents theamplification and subsequent detection of the PCR product associatedwith targeted DNA. The present invention is not limited to aconcentration of 10,000 cells/mL.

Odds ratios of antibiotic resistance in polymicrobial versusmonomicrobial specimens are shown in Table 1, along with the odds ratioof resistance for each increase in the number of bacterial species inpolymicrobial specimens. The resistance rates of polymicrobial sampleswere generally higher than the rates of monomicrobial samples: 10 of 18antibiotics had statistically higher resistance rates for polymicrobialsamples. The odds of resistance for each additional species identifiedin a polymicrobial specimen increased for ampicillin,amoxicillin/clavulanate, five of the six of the cephalosporins tested,vancomycin, and tetracycline. The opposite was true forpiperacillin/tazobactam, where each additional species in apolymicrobial specimen resulted in a 75% decrease in the odds ofresistance (95% Cl 0.61, 0.94, p=0.01). The present invention is notlimited to a concentration of 10,000 cells/mL.

Referring to Table 1, the numbers in parentheses are 95% confidenceinterval boundaries. Column 2 represents odds ratios of resistance forpolymicrobial versus monomicrobial specimens. Column 4 presents oddsratios of resistance for each additional bacterial species in aspecimen. (*Significant p-value.)

TABLE 1 Odds Ratios of Antibiotic Resistance Odds Ratio of ResistanceOdds Ratio of Polymicrobial Resistance for Each Antibiotic v.Monomicrobial p-value Additional Species p-value Penicillins Ampicillin1.37 (1.10, 1.70) 0.005* 1.14 (1.05, 1.24) 0.001* CombinationsAmoxicillin/Clavulanate 1.38 (1.09, 1.74) 0.008* 1.16 (1.07, 1.26)0.0005* Ampicillin/Sulbactam 1.13 (0.89, 1.42) 0.82 1.05 (0.96, 1.14)0.30 Trimethoprim/Sulfamethoxazole 1.09 (0.88, 1.37) 0.43 0.98 (0.91,1.07) 0.69 Piperacillin/Tazobactam 0.69 (0.43, 1.11) 0.12 0.75 (0.61,0.94) 0.010* Cephalosporins Cefaclor 1.42 (1.15, 1.77) 0.001* 1.15(1.06. 1.25) 0.0006* Cefazolin 1.38 (1.11, 1.72) 0.004* 1.15 (1.07,1.25) 0.0004* Cefepime 1.45 (1.16, 1.80) 0.001* 1.12 (1.03, 1.21) 0.006*Cefoxitin 1.41 (1.14, 1.78) 0.002* 1.10 (1.01, 1.19) 0.020* Ceftazidime1.31 (1.05, 1.62) 0.02* 1.07 (0.99, 1.15) 0.10 Ceftriaxone 1.25 (1.01,1.56) 0.04* 1.09 (1.01, 1.18) 0.03* Carbapenems Meropenem 1.28 (0.99,1.65) 0.06 1.08 (0.99, 1.18) 0.10 Aminoglycosides Gentamicin 1.17 (0.90,1.51) 0.23 1.01 (0.92, 1.11) 0.78

Ciprofloxacin 1.20 (0.96, 1.51) 0.11 1.03 (0.95, 1.12) 0.45 Levofloxacin1.20 (0.95, 1.53) 0.13 1.06 (0.97, 1.14) 0.26 Nitrofurans Nitrofurantoin1.03 (0.64, 1.85) 0.9 0.90 (0.74, 1.08) 0.25 Tetracyclines Tetracyclines1.26 (1.02, 1.57) 0.04* 1.11 (1.02, 1.20) 0.010* GlycopeptidesVancomycin 2.15 (1.63, 2.84) <0.0001 1.38 (1.21, 1.56) <0.0001

indicates data missing or illegible when filed

FIG. 2 shows the effect of specific species interactions on theprobability of increased or decreased resistance to each antibiotictested. No interactions were detected for nitrofurantoin andpiperacillin/tazobactam. Whereas the odds of resistance to ampicillin,amoxicillin/clavulanate, 6 different cephalosporins, vancomycin, andtetracycline increased with increasing number of detected species (FIG.2), there were 19 instances for which 11 of the 13 bacterial pairsresulted in reduced susceptibility to the same antibiotics.

Using HSAP, there were 44 instances for which 13 pairs of bacteriashowed statistically significant interactions that either increased ordecreased the probability of resistance to the antibiotics tested.According to the HSAP principle, most interactions resulted in adecreased probability of resistance. Only 6/44 (13.6%) pairings resultedin increased odds of antibiotic resistance, whereas a decreasedprobability occurred in 38/44 (86.4%) of pairings.

The bacterial combinations that increased the probability of antibioticresistance according to the HSAP model were E. faecalis and K.pneumoniae (amoxicillin/clavulanate, p=0.02 and ampicillin/sulbactam,p=0.03), E. coli and K. pneumoniae (ampicillin/sulbactam, p=0.04 andcefaclor, p=0.05), CoNS and E. coli and (levofloxacin, p<0.001), E.faecalis and S. agalactiae (tetracycline, p<0.001).

The UP model identified 49 statistically significant interactions, allof which showed decreased probability of resistance to the antibioticstested.

To illustrate the model, one specific pair is presented in graphicalform. FIG. 1 shows the predicted probabilities of resistance toampicillin/sulbactam, cefaclor, and tetracycline by monomicrobialpositive cultures for E. coli and K. pneumoniae and a polymicrobialculture positive for both E. coli and K. pneumoniae. When the HSAP modelwas used, the pairing of E. coli and K. pneumoniae resulted in either asignificant increase or significant decrease in the probability ofresistance depending on the antibiotic tested. For example, whenampicillin/sulbactam or cefaclor was applied to the combination of E.coli and K. pneumoniae, the resistance rate was higher than either E.coli or K. pneumoniae alone. In contrast, the resistance rate totetracycline of same combination of species, E. coli and K. pneumoniae,was intermediate between the resistance rates to each species alone.

These results demonstrate that polymicrobial infections, whichconstituted 56.1% (758/1,352) of positive samples with susceptibilityresults, can alter response to antibiotics. They also show that thealteration is sensitive to both the specific bacterial combination andthe antibiotic tested. Thirteen bacterial pairs had one or moresignificant interactions when tested on 16 of the 18 antibiotics usingHSAP and UP. Of these interactions, 38 resulted in a decreasedprobability of resistance, while 6 resulted in an increased probabilityof resistance. The combination of E. coli and K. pneumoniae resulted inan increased probability of resistance to ampicillin/sulbactam andcefaclor, but decreased probability of resistance to tetracycline. E.faecalis together with K. pneumoniae resulted in increased resistance toamoxicillin/clavulanate and ampicillin/sulbactam, but decreasedresistance to levofloxacin, meropenem, and tetracycline. E. faecaliscombined with S. agalactiae produced an increase in resistance totetracycline, but decreased resistance to ampicillin and vancomycin.Similarly, the combination of CoNS and E. coli produced an increasedprobability in resistance to levofloxacin, but the same combinationproduced a decreased probability in resistance toamoxicillin/clavulanate, ceftriaxone, tetracycline, andtrimethoprim/sulfamethoxazole. These differences may be attributed tothe unique mechanisms of action of the specific antibiotics.

A similar set of contrasts is observed from the perspective ofindividual antibiotics. Different pairs of bacteria caused bothincreased and decreased resistance to amoxicillin/clavulanate,ampicillin/sulbactam, cefaclor, levofloxacin, and tetracycline. Forinstance, E. coli combined with K. pneumoniae produced an increase inresistance to cefaclor, while E. coli combined with P. mirabilisproduced a decrease in resistance to cefaclor. These results highlightthe importance of accurate identification of bacteria in polymicrobialinfections: a difference in identification of one species can influenceantibiotic resistance.

The observed effects on antibiotic resistance in polymicrobialinfections may be due to cooperative and/or competitive interactionsbetween bacteria. Resistant bacteria can cooperatively protectsusceptible bacteria by degrading antibiotics, as occurs when secretedbeta-lactamase degrades beta-lactam antibiotics. Antibiotic resistancecan be conferred by one bacterium on another bacterium by means ofhorizontal gene transfer (HGT) of antibiotic resistance genes. Bacterialinteractions with host macrophages can promote HGT. For instance, P.aeruginosa, when present in biofilms, produces extracellular DNA thatinduces neutrophils to produce pro-inflammatory cytokines (IL-8 and IL-1beta). The ensuing inflammation can promote HGT involving E. coli.Interestingly, some antibiotics can also promote HGT: antibiotics thatcause bacterial lysis release DNA and proteins that can be taken up byother bacteria. In addition, one bacterium can stimulate gene expressionin another bacterium, resulting in upregulation of efflux pumps leadingto increased antibiotic resistance. Bacterial community spatialstructuring within a polymicrobial biofilm may also affect the efficacyof antibiotics.

Decreased resistance to antibiotics in polymicrobial specimens may alsobe due to competitive mechanisms between bacteria. P. aeruginosa hasbeen documented to produce antibiotics, whereas Enterococcus speciesproduce and secrete bacteriocins. Gram-negative bacteria have developeda number of specialized secretion systems that can perform protectivefunctions. Type V secretion systems secrete proteases that digest IgA,surface receptors that bind the constant region of IgG, and virulencefactor/adhesin proteins that promote colonization. Type VI secretionsystems allow Gram-negative bacteria to secrete antibacterial toxinsdirectly into other bacteria. At the same time, Type VI systems mediateDNA acquisition via HGT; an example is the capacity for A. baumannii torapidly acquire resistance genes from E. coli by means of Type VItransfer systems.

One type of bacterial interaction can cause a paradoxical result:cross-feeding between bacteria can produce decreased antibioticresistance. This may explain our observed decreased probability ofantibiotic resistance seen in most specific organism combinations.Cross-feeding is a process by which one organism produces metabolitesthat promote the survival of another organism. However, this interactioncan produce a chain of dependencies, leaving the entire chain only asresistant as the most susceptible bacterium. Adamowicz et al. showedthat bacterial species were inhibited at significantly lower antibioticconcentrations in cross-feeding communities than in monoculture; coinedas the “weakest link” model.

Example 2. Antibiotic Resistance (ABR) Assay Utilizing Agar-ContainingMedium

Urine samples suitable for processing with this assay are collected,transported, and stored using BD Vacutainer (gray top) tubes or othersuitable leak-proof sterile container. Urine samples may be held at roomtemperature for 48 hours before test results are compromised.

Antibiotics not received in ready-made solutions were dissolved inappropriate solvent and according to their individual solubility at 10×the concentration desired in the assay as antibiotic stocks. Antibioticstocks are stored at 2-8° C. and protected from direct sunlight.Prepared antibiotic stock solutions were aliquoted into a 96-deep wellplate (Thermo Fisher Scientific) to form an Antibiotic Source Plate, asshown in FIG. 11 and identified by antibiotic name and concentration(μg/mL; 10× final concentration). Antibiotics include in this assay werenitrofurantoin, ciprofloxacin, meropenem, ceftriaxone, trimethoprim,sulfamethoxazole; piperacillin, tazobactam, levofloxacin, cefoxitin,tetracycline, ampicillin, sulbactam, and vancomycin, either singly or incombination. One well was designated AB-blend which contained acombination of antibiotics to ensure there was no bacterial growth.

One hundred microliters of Mueller-Hinton agar medium was aliquoted intoeach appropriate well position of a 96-well microplate (VIS 96/F-PS,Eppendorf). The medium was allowed to solidify at room temperature forat least 10 min.

The antibiotics (10 μL) at various concentrations were then aliquotedinto desired wells from the Antibiotic Source Plate. After theantibiotics were introduced to the agar medium, the ABR microplates wereallowed to sit for at least 1 hr before use. If long-term storage isrequired, ABR microplates containing antibiotic-infuse agar are storedat 2-8° C. in the dark.

At the time of testing, urine samples were diluted 1:20 in sterilesaline and vortexed. Each patient sample utilized a single ABRmicroplate. Five microliters of diluted patient sample were added toeach well of the room temperature microplate, the plate was sealed andincubated for 16-18 hr at 37° C.

After incubation, the plate was removed from the incubator and carefullyuncovered. Two-hundred microliters of deionized water were added to eachwell to suspend cells present above the agar and the plates incubated atroom temperature for 30 min. After 30 min, 100 μl from each well wasremoved to a new plate and the OD₆₀₀ was determined in aspectrophotometer. Five separate reads were taken of each plate and amean OD₆₀₀ measurement calculated.

Controls include: No-antibiotic control, Negative control plate, andAB-Blend. No antibiotic control: Any well containing medium that is notinfused with antibiotics to ensure viability of bacterial cells presentin patient urine samples and included in each plate. If theno-antibiotic control for any given patient does not yield growth, asecondary test is performed using the same patient sample withoutdilution. Negative control plate: Microplate containingantibiotic-infused agar medium without addition of patient sample orcultured bacterial organisms to ensure non-contamination of reagents.AB-Blend: One or more wells containing a combination of antibiotics toensure there is no bacterial growth.

Raw data collected from the plate is depicted in Table 2. Data inspreadsheet form was arranged as “Well Position” adjacent to itscorresponding “Mean” OD.

TABLE 2 Well Mean A7 0.6960 A8 0.0388 A9 0.0744 A10 0.0385 A11 0.0477A12 0.4550 B7 0.0387 B8 0.0390 B9 0.0412 B10 0.4250 B11 0.0449 B120.4880 C7 0.0386 C8 0.0385 C9 0.0445 C10 0.4296 C11 0.0401 C12 0.5222 D70.0392 D8 0.5372 D9 0.0432 D10 0.4377 D11 0.0392 D12 0.4824 E7 0.0408 E80.5029 E9 0.0405 E10 0.4918 E11 0.0389 E12 0.5087 F7 0.0414 F8 0.2925 F90.0392 F10 0.4378 F11 0.0389 F12 0.5307 G7 0.0387 G8 0.0408 G9 0.0391G10 0.0495 G11 0.4304 G12 0.0384 H7 0.0392 H8 0.0401 H9 0.0386 H100.0474 H11 0.4396 H12 0.7874

Each well position corresponds to a particular antibiotic at a certainconcentration according to the plate plan. Addition of the antibioticlegend is depicted in Table 3.

TABLE 3 Antibiotic Well Mean No-antibiotic A7 0.6960 Mero-2 A8 0.0388Levo-4 A9 0.0744 Ceftria-64 A10 0.0385 Pip/Tazo-64, 4 A11 0.0477Tetra-16 A12 0.4550 Nitro-32 B7 0.0387 Mero-4 B8 0.0390 Levo-8 B9 0.0412Vanco-2 B10 0.4250 Pip/Tazo-128, 4 B11 0.0449 Amp-8 B12 0.4880 Nitro-64C7 0.0386 Mero-8 C8 0.0385 Ceftria-1 C9 0.0445 Vanco-4 C10 0.4296Cefox-4 C11 0.0401 Amp-16 C12 0.5222 Nitro-128 D7 0.0392 Amp/Sulb-8, 4D8 0.5372 Ceftria-2 D9 0.0432 Vanco-8 D10 0.4377 Cefox-8 D11 0.0392Amp-32 D12 0.4824 Cipro-1 E7 0.0408 Amp/Sulb-16, 8 E8 0.5029 Ceftria-4E9 0.0405 Vanco-16 E10 0.4918 Cefox-16 E11 0.0389 TMP/SMX-2, 38 E120.5087 Cipro-2 F7 0.0414 Amp/Sulb-32, 16 F8 0.2925 Ceftria-8 F9 0.0392Vanco-32 F10 0.4378 Cefox-32 F11 0.0389 TMP/SMX-4, 76 F12 0.5307 Cipro-4G7 0.0387 Levo-1 G8 0.0408 Ceftria-16 G9 0.0391 Pip/Tazo-16, 4 G100.0495 Tetra-4 G11 0.4304 AB-Blend G12 0.0384 Mero-1 H7 0.0392 Levo-2 H80.0401 Ceftria-32 H9 0.0386 Pip/Tazo-32, 4 H10 0.0474 Tetra-8 H11 0.4396empty H12 0.7874

Once the antibiotic legend was placed adjacent to the appropriate well,the data was rearranged by sorting like antibiotics together (Table 4).

TABLE 4 Antibiotic Well Mean No-antibiotic A7 0.6960 Nitro-32 B7 0.0387Nitro-64 C7 0.0386 Nitro-128 D7 0.0392 Cipro-1 E7 0.0408 Cipro-2 F70.0414 Cipro-4 G7 0.0387 Mero-1 H7 0.0392 Mero-2 A8 0.0388 Mero-4 B80.0390 Mero-8 C8 0.0385 Amp/Sulb-8, 4 D8 0.5372 Amp/Sulb-16, 8 E8 0.5029Amp/Sulb-32, 16 F8 0.2925 Levo-1 G8 0.0408 Levo-2 H8 0.0401 Levo-4 A90.0744 Levo-8 B9 0.0412 Ceftria-1 C9 0.0445 Ceftria-2 D9 0.0432Ceftria-4 E9 0.0405 Ceftria-8 F9 0.0392 Ceftria-16 G9 0.0390 Ceftria-32H9 0.0386 Ceftria-64 A10 0.0385 Vanco-2 B10 0.4250 Vanco-4 C10 0.4296Vanco-8 D10 0.4377 Vanco-16 E10 0.4918 Vanco-32 F10 0.4378 Pip/Tazo-16,4 G10 0.0495 Pip/Tazo-32, 4 H10 0.0474 Pip/Tazo-64, 4 A11 0.0477Pip/Tazo-128, 4 B11 0.0449 Cefox-4 C11 0.0401 Cefox-8 D11 0.0392Cefox-16 E11 0.0389 Cefox-32 F11 0.0389 Tetra-4 G11 0.4304 Tetra-8 H110.4396 Tetra-16 A12 0.4550 Amp-8 B12 0.4880 Amp-16 C12 0.5222 Amp-32 D120.4824 TMP/SMX-2, 38 E12 0.5087 TMP/SMX-4, 76 F12 0.5307 AB-Blend G120.0384 empty H12 0.7874

The raw data was then “blanked” using the measurement obtained from theAB-Blend well, as depicted in Table 5.

TABLE 5 Antibiotic Well Mean Blanked No-antibiotic A7 0.6960 0.6576Nitro-32 B7 0.0387 0.0003 Nitro-64 C7 0.0386 0.0002 Nitro-128 D7 0.03920.0008 Cipro-1 E7 0.0408 0.0024 Cipro-2 F7 0.0414 0.0030 Cipro-4 G70.0387 0.0003 Mero-1 H7 0.0392 0.0008 Mero-2 A8 0.0388 0.0004 Mero-4 B80.0390 0.0006 Mero-8 C8 0.0385 0.0001 Amp/Sulb-8,4 D8 0.5372 0.4988Amp/Sulb-16,8 E8 0.5029 0.4646 Amp/Sulb-32,16 F8 0.2925 0.2541 Levo-1 G80.0408 0.0024 Levo-2 H8 0.0401 0.0017 Levo-4 A9 0.0744 0.0360 Levo-8 B90.0412 0.0028 Ceftria-1 C9 0.0445 0.0061 Ceftria-2 D9 0.0432 0.0048Ceftria-4 E9 0.0405 0.0021 Ceftria-8 F9 0.0392 0.0008 Ceftria-16 G90.0391 0.0007 Ceftria-32 H9 0.0386 0.0002 Ceftria-64 A10 0.0385 0.0001Vanco-2 B10 0.4250 0.3866 Vanco-4 C10 0.4296 0.3912 Vanco-8 D10 0.43770.3993 Vanco-16 E10 0.4918 0.4534 Vanco-32 F10 0.4378 0.3994Pip/Tazo-16,4 G10 0.0495 0.0111 Pip/Tazo-32,4 H10 0.0474 0.0090Pip/Tazo-64,4 A11 0.0477 0.0093 Pip/Tazo-128,4 B11 0.0449 0.0065 Cefox-4C11 0.0401 0.0017 Cefox-8 D11 0.0392 0.0008 Cefox-16 E11 0.0389 0.0005Cefox-32 F11 0.0389 0.0005 Tetra-4 G11 0.4304 0.3920 Tetra-8 H11 0.43960.4012 Tetra-16 A12 0.4550 0.4166 Amp-8 B12 0.4880 0.4496 Amp-16 C120.5222 0.4838 Amp-32 D12 0.4824 0.4440 TMP/SMX-2,38 E12 0.5087 0.4703TMP/SMX-4,76 F12 0.5307 0.4923 AB-Blend G12 0.0384 0 empty H12 0.78740.7490

To determine whether bacterial organisms present in the patient sampleswere resistant or sensitive to a particular antibiotic at a certainconcentration, blanked OD readings were compared to a threshold OD₆₀₀ of0.025 (Table 6). Any OD measurement greater than or equal to thisthreshold was designated Resistant (R) meaning bacterial organismspresent in patient sample were resistant to that particular antibioticat that certain concentration. Any OD measurement less than thisthreshold was designated Sensitive (S) meaning bacterial organismspresent in patient sample were sensitive to that particular antibioticat that certain concentration.

TABLE 6 Antibiotic Well Mean Blanked Result No-antibiotic A7 0.69600.6576 R Nitro-32 B7 0.0387 0.0003 S Nitro-64 C7 0.0386 0.0002 SNitro-128 D7 0.0392 0.0008 S Cipro-1 E7 0.0408 0.0024 S Cipro-2 F70.0414 0.0030 S Cipro-4 G7 0.0387 0.0003 S Mero-1 H7 0.0392 0.0008 SMero-2 A8 0.0388 0.0004 S Mero-4 B8 0.0390 0.0006 S Mero-8 C8 0.03850.0001 S Amp/Sulb-8,4 D8 0.5372 0.4988 R Amp/Sulb-16,8 E8 0.5029 0.4646R Amp/Sulb-32,16 F8 0.2925 0.2541 R Levo-1 G8 0.0408 0.0024 S Levo-2 H80.0401 0.0017 S Levo-4 A9 0.0744 0.0360 R Levo-8 B9 0.0412 0.0028 SCeftria-1 C9 0.0445 0.0061 S Ceftria-2 D9 0.0432 0.0048 S Ceftria-4 E90.0405 0.0021 S Ceftria-8 F9 0.0392 0.0008 S Ceftria-16 G9 0.0391 0.0007S Ceftria-32 H9 0.0386 0.0002 S Ceftria-64 A10 0.0385 0.0001 S Vanco-2B10 0.4250 0.3866 R Vanco-4 C10 0.4296 0.3912 R Vanco-8 D10 0.43770.3993 R Vanco-16 E10 0.4918 0.4534 R Vanco-32 F10 0.4378 0.3994 RPip/Tazo-16,4 G10 0.0495 0.0111 S Pip/Tazo-32,4 H10 0.0474 0.0090 SPip/Tazo-64,4 A11 0.0477 0.0093 S Pip/Tazo-128,4 B11 0.0449 0.0065 SCefox-4 C11 0.0401 0.0017 S Cefox-8 D11 0.0392 0.0008 S Cefox-16 E110.0389 0.0005 S Cefox-32 F11 0.0389 0.0005 S Tetra-4 G11 0.4304 0.3920 RTetra-8 H11 0.4396 0.4012 R Tetra-16 A12 0.4550 0.4166 R Amp-8 B120.4880 0.4496 R Amp-16 C12 0.5222 0.4838 R Amp-32 D12 0.4824 0.4440 RTMP/SMX-2,38 E12 0.5087 0.4703 R TMP/SMX-4,76 F12 0.5307 0.4923 RAB-Blend G12 0.0384 0 S empty H12 0.7874 0.7490 R

In this example, the sample contains bacteria sensitive tonitrofurantoin, ciprofloxacin, meropenem, ceftriaxone,piperacillin/tazobactam, and cefoxitin. The results for levo areequivocal.

The MIC for each drug can then be provided. The minimum inhibitoryconcentration (MIC) is the minimum test antibiotic concentration towhich the sample is sensitive. An exemplary MIC determination formeropenem based on the results above is depicted in Table 7.

TABLE 7 Mero Mero Mero Mero Inter- 113 [1] 114 [2] 115 [4] 116 [8] 117MIC 118 pretation 119 S 120 S 121 S 122 S 123 <=1 124 S 125 R 126 S 127S 128 S 129 <=2 130 I 131 R 132 R 133 S 134 S 135 <=4 136 I 137 R 138 R139 R 140 S 141 <=8 142 I 143 R 144 R 145 R 146 R 147 >=8 148 R

Example 3. Validation of ABR Assay Utilizing Agar Containing GrowthMedium

Accuracy

Accuracy was assessed by comparing the antibiotic resistance results ofthe test method compared to those obtained for mixed and isolatedcultures evaluated by the antibiotic-agar method. A total of 19bacterial pools (pools consist of 2-4 organisms), 17 isolated organisms,and 9 routinely processed urine samples were tested for resistance to 12antibiotics. Accuracy was assessed in regards to Specificity (TrueNegatives), Sensitivity (True Positives), and Overall Accuracy (AllSamples). The assay showed good accuracy in all three categories (Table8).

TABLE 8 % Accuracy Overall 96% Accuracy Specificity 95% Sensitivity 96%

Precision

Inter-assay precision was evaluated by testing three samples from the“Accuracy” sample set over three days. Intra-assay precision wasevaluated by testing each of these samples in triplicate in one batch.Precision for each sample was assessed by determining the consensusresult of all 5 replicates and then counting the number of replicatesthat match the consensus. This number was then divided by the sum of allmeasurements (sum of measurements for all drugs) to determine the %precision. The overall precision was calculated by dividing the sum ofall correct matches by the total number of measurements from allsamples. The assay demonstrated very good precision (Table 9).

TABLE 9 All Precision Samples Total Matched 643 Total 690 Measurements %Match 93%

Analytical Sensitivity

Analytic sensitivity, or the limit of detection (LOD), was assessed bydetermined the lowest bacterial concentration that yielded accurateresults. In certain cases, bacterial concentrations lower than 10,000cells/mL are not considered positive for UTI and therefore the lowestconcentration tested was 10,000 cells/mL. Consistent results (>96%)correlation to the consensus results were obtained at the lowestbacterial concentrations tested. The LOD of this assay was 10,000cells/mL. Note, the present invention is not limited to a concentrationof 10,000 cells/mL.

Analytical Specificity

The analytic specificity of this assay was assessed by testing samplesat bacterial concentrations of 100,000,000 cells/mL. Such concentrationsare not typically observed in routine UTI patient samples but wereachieved in saturated overnight bacterial cultures. Assessment ofanalytic measurement range (AMR) was then performed by testing threesamples from the “Accuracy” sample set each diluted as follows:100,000,000 cells/mL, 1,000,000 cells/mL, 100,000 cells/mL and 10,000cells/mL. Consistent results (>94%) correlation to the consensus resultswere obtained at all bacterial concentrations tested. The assay isspecific at bacterial concentration up to 100,000,000 cells/mL. Thepresent invention is not limited to a concentration of 10,000 cells/mL.

Example 4. Antibiotic Resistance (ABR) Assay Utilizing Liquid GrowthMedium

Urine samples suitable for processing with this assay are collected,transported, and stored using BD Vacutainer tubes or other suitableleak-proof sterile containers. Urine samples may be held at roomtemperature for 48 hours before test results are compromised.

Antibiotics not received in ready-made solutions were dissolved inappropriate solvents and according to their individual solubility to 50×the concentration desired in the assay and stored as antibiotic stocks.Antibiotic stocks are stored at 2-8° C. and protected from directsunlight. Prepared antibiotic stock solutions were aliquoted into a96-deep well plate (ThermoFisher Scientific) to form a 50× AntibioticSource Plate and then diluted 1:5 to form a 10× Antibiotic Source Plate,as shown in FIG. 12 where each well is identified by antibiotic name andconcentration (μg/mL; 10× final concentration). Antibiotics included inthis assay were amoxicillin, clavulanate, ampicillin, sulbactam,cefaclor, cefazolin, cefepime, cefoxitin, ceftazidime, ceftriaxone,ciprofloxacin, gentamicin, levofloxacin, meropenem, nitrofurantoin,piperacillin, tazobactam, tetracycline, trimethoprim, sulfamethoxazole,and vancomycin, either singly or in combination. One well was assignedsodium azide to ensure no bacterial growth would be observed in thatwell.

Twenty microliters of each antibiotic solution were aliquoted into thepre-determined wells of a 96-well microplate (VIS 96/F-PS, Eppendorf)from the 10× Antibiotic Source Plate to create ABR testing plates forinoculation. These ABR testing plates were allowed to sit for up to 24hours before use at 2-8° C. in the dark.

At the time of testing, urine samples were centrifuged to concentrateany bacterial cells and then mixed with liquid Mueller-Hinton medium andincubated for 6-16 hours at 37° C. After this initial incubation, thesample is diluted to 0.5-0.6 McF in saline and then 500 μl of thatsuspension was added to 29.5 μl of Mueller-Hinton medium. One-hundredand eighty microliters of the diluted sample is then aliquoted to eachwell of the ABR microplate already containing 10× antibiotic solution,bringing all of the antibiotics to the desired final concentration. Theplate is then sealed and incubated for 12-16 hours at 37° C.

After incubation, the plate was removed from the incubator and carefullyuncovered and the OD600 was determined for each appropriate well byspectrophotometer. Five separate measurements were taken of each well ona plate and the mean OD600 measurement calculated for each well.

Controls are depicted in Table 10.

TABLE 10 Control Name Control Conditions No-antibiotic control Any wellcontaining medium that is not infused with antibiotics to ensureviability of bacterial cells present in patient urine samples andincluded in each plate. If the no-antibiotic control for any givenpatient does not yield growth, the sample is repeated on the assay andreported as quantity not sufficient if repeat testing still does notyield satisfactory results. Negative control plate Microplate containingantibiotic-infused agar medium without addition of patient sample orcultured bacterial organisms to ensure non-contamination of reagents. NaAzide One or more wells containing a dilute concentration of sodiumazide to ensure no bacterial growth will occur.

Raw data collected from the plate is depicted in Table 11. Data inspreadsheet form was arranged as “Well Position” adjacent to itscorresponding “Mean” OD.

TABLE 11 Well Mean A1 0.2217 A2 0.0496 A3 0.2357 A4 0.0421 A5 0.0539 A60.1468 A7 0.2457 A8 0.0427 B1 0.0552 B2 0.0413 B3 0.2449 B4 0.0419 B50.0417 B6 0.0570 B7 0.2607 B8 0.0419 C1 0.0539 C2 0.0414 C3 0.2356 C40.2202 C5 0.0419 C6 0.2416 C7 0.2473 C8 0.2332 D1 0.0441 D2 0.1180 D30.0504 D4 0.2288 D5 0.0418 D6 0.2427 D7 0.2437 D8 0.0600 E1 0.0423 E20.0436 E3 0.0435 E4 0.2348 E5 0.1209 E6 0.2417 E7 0.0615 E8 0.0457 F10.2198 F2 0.0431 F3 0.0425 F4 0.2084 F5 0.1016 F6 0.2404 F7 0.0426 F80.2604 G1 0.1928 G2 0.0443 G3 0.0431 G4 0.2224 G5 0.2339 G6 0.2323 G70.0418 G8 0.2354 H1 0.1556 H2 0.2485 H3 0.0426 H4 0.1596 H5 0.2090 H60.2281 H7 0.0437 H8 0.2446

Each well position corresponds to a particular antibiotic at a certainconcentration according to the plate plan. Addition of the antibioticlegend is depicted in Table 12.

TABLE 12 Antibiotic Well Mean No-Antibiotic A1 0.2217 Mero-8 A2 0.0496Ceftriaxone-4 A3 0.2357 Pip/Tazo-16,4 A4 0.0421 Tetra-16 A5 0.0539Cefazolin-16 A6 0.1468 Ceftazidime-4 A7 0.2457 No-Antibiotic A8 0.0427Nitro-32 B1 0.0552 Amp/Sulb-8,4 B2 0.0413 Ceftriaxone-8 B3 0.2449Pip/Tazo-128,4 B4 0.0419 Amp-8 B5 0.0417 Cefazolin-32 B6 0.0570Ceftazidime-8 B7 0.2607 No-Antibiotic B8 0.0419 Nitro-128 C1 0.0539Amp/Sulb-32,16 C2 0.0414 Ceftriaxone-64 C3 0.2356 Cefoxitin-4 C4 0.2202Amp-16 C5 0.0419 Cefepime-1 C6 0.2416 Ceftazidime-16 C7 0.2473Cefaclor-8 C8 0.2332 Cipro-1 D1 0.0441 Levo-1 D2 0.1180 Vanco-1 D30.0504 Cefoxitin-8 D4 0.2288 Amp-32 D5 0.0418 Cefepime-2 D6 0.2427Ceftazidime-32 D7 0.2437 Cefaclor-32 D8 0.0600 Cipro-4 E1 0.0423 Levo-2E2 0.0436 Vanco-2 E3 0.0435 Cefoxitin-32 E4 0.2348 TMP/SMX-2,38 E50.1209 Cefepime-4 E6 0.2417 Gentamicin-4 E7 0.0615 Na Azide E8 0.0457Mero-1 F1 0.2198 Levo-4 F2 0.0431 Vanco-4 F3 0.0425 Tetra-2 F4 0.2084TMP/SMX-4,76 F5 0.1016 Cefepime-8 F6 0.2404 Gentamicin-16 F7 0.0426No-Antibiotic F8 0.2604 Mero-2 G1 0.1928 Levo-8 G2 0.0443 Vanco-16 G30.0431 Tetra-4 G4 0.2224 Cefazolin-2 G5 0.2339 Cefepime-16 G6 0.2323Amox/Clav-8,4 G7 0.0418 Ceftriaxone-8 B3 0.2449 No-Antibiotic G8 0.2354Mero-4 H1 0.1556 Ceftriaxone-1 H2 0.2485 Vanco-32 H3 0.0426 Tetra-8 H40.1596 Cefazolin-8 H5 0.2090 Cefepime-32 H6 0.2281 Amox/Clav-32,16 H70.0437 No-Antibiotic H8 0.2446

With antibiotic legend placed adjacent to the appropriate well, the datawas rearranged by sorting like antibiotics together (Table 13).

TABLE 13 Antibiotic Well Mean Na Azide E8 0.0457 No-Antibiotic A1 0.2217No-Antibiotic F8 0.2604 No-Antibiotic G8 0.2354 No-Antibiotic H8 0.2446Amox/Clav-8,4 G7 0.0418 Amox/Clav-32,16 H7 0.0437 Amp-8 B5 0.0417 Amp-16C5 0.0419 Amp-32 D5 0.0418 Amp/Sulb-8,4 B2 0.0413 Amp/Sulb-32,16 C20.0414 Cefaclor-8 C8 0.2332 Cefaclor-32 D8 0.0600 Cefazolin-2 G5 0.2339Cefazolin-8 H5 0.2090 Cefazolin-16 A6 0.1468 Cefazolin-32 B6 0.0570Cefepime-1 C6 0.2416 Cefepime-2 D6 0.2427 Cefepime-4 E6 0.2417Cefepime-8 F6 0.2404 Cefepime-16 G6 0.2323 Cefepime-32 H6 0.2281Cefoxitin-4 C4 0.2202 Cefoxitin-8 D4 0.2288 Cefoxitin-32 E4 0.2348Ceftazidime-4 A7 0.2457 Ceftazidime-8 B7 0.2607 Ceftazidime-16 C7 0.2473Ceftazidime-32 D7 0.2437 Ceftriaxone-1 H2 0.2485 Ceftriaxone-4 A3 0.2357Ceftriaxone-8 B3 0.2449 Ceftriaxone-64 C3 0.2356 Cipro-1 D1 0.0441Cipro-4 E1 0.0423 No-Antibiotic A8 0.0427 No-Antibiotic B8 0.0419Gentamicin-4 E7 0.0615 Gentamicin-16 F7 0.0426 Levo-1 D2 0.1180 Levo-2E2 0.0436 Levo-4 F2 0.0431 Levo-8 G2 0.0443 Mero-1 F1 0.2198 Mero-2 G10.1928 Mero-4 H1 0.1556 Mero-8 A2 0.0496 Nitro-32 B1 0.0552 Nitro-128 C10.0539 Pip/Tazo-16,4 A4 0.0421 Pip/Tazo-128,4 B4 0.0419 Tetra-2 F40.2084 Tetra-4 G4 0.2224 Tetra-8 H4 0.1596 Tetra-16 A5 0.0539TMP/SMX-2,38 E5 0.1209 TMP/SMX-4,76 F5 0.1016 Vanco-1 D3 0.0504 Vanco-2E3 0.0435 Vanco-4 F3 0.0425 Vanco-16 G3 0.0431 Vanco-32 H3 0.0426

The raw data was then “blanked” using the measurement obtained from theNa-Azide well, as depicted in Table 14.

TABLE 14 Antibiotic Well Mean Blanked Na Azide E8 0.0457 0 No-AntibioticA1 0.2217 0.1760 No-Antibiotic F8 0.2604 0.2147 No-Antibiotic G8 0.23540.1897 No-Antibiotic H8 0.2446 0.1989 Amox/Clav-8,4 G7 0.0418 −0.0039Amox/Clav-32,16 H7 0.0437 −0.0020 Amp-8 B5 0.0417 −0.0040 Amp-16 C50.0419 −0.0038 Amp-32 D5 0.0418 −0.0039 Amp/Sulb-8,4 B2 0.0413 −0.0044Amp/Sulb-32,16 C2 0.0414 −0.0043 Cefaclor-8 C8 0.2332 0.1875 Cefaclor-32D8 0.0600 0.0143 Cefazolin-2 G5 0.2339 0.1882 Cefazolin-8 H5 0.20900.1633 Cefazolin-16 A6 0.1468 0.1011 Cefazolin-32 B6 0.0570 0.0113Cefepime-1 C6 0.2416 0.1959 Cefepime-2 D6 0.2427 0.1970 Cefepime-4 E60.2417 0.1960 Cefepime-8 F6 0.2404 0.1947 Cefepime-16 G6 0.2323 0.1866Cefepime-32 H6 0.2281 0.1824 Cefoxitin-4 C4 0.2202 0.1745 Cefoxitin-8 D40.2288 0.1831 Cefoxitin-32 E4 0.2348 0.1891 Ceftazidime-4 A7 0.24570.2000 Ceftazidime-8 B7 0.2607 0.2150 Ceftazidime-16 C7 0.2473 0.2016Ceftazidime-32 D7 0.2437 0.1980 Ceftriaxone-1 H2 0.2485 0.2028Ceftriaxone-4 A3 0.2357 0.1900 Ceftriaxone-8 B3 0.2449 0.1992Ceftriaxone-64 C3 0.2356 0.1899 Cipro-1 D1 0.0441 −0.0016 Cipro-4 E10.0423 −0.0034 No-Antibiotic A8 0.0427 −0.0030 No-Antibiotic B8 0.0419−0.0038 Gentamicin-4 E7 0.0615 0.0158 Gentamicin-16 F7 0.0426 −0.0031Levo-1 D2 0.1180 0.0723 Levo-2 E2 0.0436 −0.0021 Levo-4 F2 0.0431−0.0026 Levo-8 G2 0.0443 −0.0014 Mero-1 F1 0.2198 0.1741 Mero-2 G10.1928 0.1471 Mero-4 H1 0.1556 0.1099 Mero-8 A2 0.0496 0.0039 Nitro-32B1 0.0552 0.0095 Nitro-128 C1 0.0539 0.0082 Pip/Tazo-16,4 A4 0.0421−0.0036 Pip/Tazo-128,4 B4 0.0419 −0.0038 Tetra-2 F4 0.2084 0.1627Tetra-4 G4 0.2224 0.1767 Tetra-8 H4 0.1596 0.1139 Tetra-16 A5 0.05390.0082 TMP/SMX-2,38 E5 0.1209 0.0752 TMP/SMX-4,76 F5 0.1016 0.0559Vanco-1 D3 0.0504 0.0047 Vanco-2 E3 0.0435 −0.0022 Vanco-4 F3 0.0425−0.0032 Vanco-16 G3 0.0431 −0.0026 Vanco-32 H3 0.0426 −0.0031

To determine whether bacterial organisms present in the patient sampleswere resistant or sensitive to a particular antibiotic at a certainconcentration, blanked OD readings were compared to a threshold OD₆₀₀ of0.065 (Table 15). An OD measurement greater than or equal to thisthreshold was designated Resistant (R) meaning bacterial organismspresent in patient sample were resistant to that particular antibioticat that certain concentration. Any OD measurement less than thisthreshold was designated Sensitive (S) meaning bacterial organismspresent in patient sample were sensitive to that particular antibioticat that certain concentration.

TABLE 15 Antibiotic Well Mean Blanked Result Na Azide E8 0.0457 0 SNo-Antibiotic A1 0.2217 0.1760 R No-Antibiotic F8 0.2604 0.2147 RNo-Antibiotic G8 0.2354 0.1897 R No-Antibiotic H8 0.2446 0.1989 RAmox/Clav-8,4 G7 0.0418 −0.0039 S Amox/Clav-32,16 H7 0.0437 −0.0020 SAmp-8 B5 0.0417 −0.0040 S Amp-16 C5 0.0419 −0.0038 S Amp-32 D5 0.0418−0.0039 S Amp/Sulb-8,4 B2 0.0413 −0.0044 S Amp/Sulb-32,16 C2 0.0414−0.0043 S Cefaclor-8 C8 0.2332 0.1875 R Cefaclor-32 D8 0.0600 0.0143 SCefazolin-2 G5 0.2339 0.1882 R Cefazolin-8 H5 0.2090 0.1633 RCefazolin-16 A6 0.1468 0.1011 R Cefazolin-32 B6 0.0570 0.0113 SCefepime-1 C6 0.2416 0.1959 R Cefepime-2 D6 0.2427 0.1970 R Cefepime-4E6 0.2417 0.1960 R Cefepime-8 F6 0.2404 0.1947 R Cefepime-16 G6 0.23230.1866 R Cefepime-32 H6 0.2281 0.1824 R Cefoxitin-4 C4 0.2202 0.1745 RCefoxitin-8 D4 0.2288 0.1831 R Cefoxitin-32 E4 0.2348 0.1891 RCeftazidime-4 A7 0.2457 0.2000 R Ceftazidime-8 B7 0.2607 0.2150 RCeftazidime-16 C7 0.2473 0.2016 R Ceftazidime-32 D7 0.2437 0.1980 RCeftriaxone-1 H2 0.2485 0.2028 R Ceftriaxone-4 A3 0.2357 0.1900 RCeftriaxone-8 B3 0.2449 0.1992 R Ceftriaxone-64 C3 0.2356 0.1899 RCipro-1 D1 0.0441 −0.0016 S Cipro-4 E1 0.0423 −0.0034 S No-Antibiotic A80.0427 −0.0030 S No-Antibiotic B8 0.0419 −0.0038 S Gentamicin-4 E70.0615 0.0158 S Gentamicin-16 F7 0.0426 −0.0031 S Levo-1 D2 0.11800.0723 R Levo-2 E2 0.0436 −0.0021 S Levo-4 F2 0.0431 −0.0026 S Levo-8 G20.0443 −0.0014 S Mero-1 F1 0.2198 0.1741 R Mero-2 G1 0.1928 0.1471 RMero-4 H1 0.1556 0.1099 R Mero-8 A2 0.0496 0.0039 S Nitro-32 B1 0.05520.0095 S Nitro-128 C1 0.0539 0.0082 S Pip/Tazo-16,4 A4 0.0421 −0.0036 SPip/Tazo-128,4 B4 0.0419 −0.0038 S Tetra-2 F4 0.2084 0.1627 R Tetra-4 G40.2224 0.1767 R Tetra-8 H4 0.1596 0.1139 R Tetra-16 A5 0.0539 0.0082 STMP/SMX-2,38 E5 0.1209 0.0752 R TMP/SMX-4,76 F5 0.1016 0.0559 S Vanco-1D3 0.0504 0.0047 S Vanco-2 E3 0.0435 −0.0022 S Vanco-4 F3 0.0425 −0.0032S Vanco-16 G3 0.0431 −0.0026 S Vanco-32 H3 0.0426 −0.0031 S

In this example, the sample contains bacteria sensitive toamoxicillin/clavulanate, ampicillin, ampicillin/sulbactam,ciprofloxacin, gentamicin, levofloxacin, nitrofurantoin,piperacillin/tazobactam, and vancomycin.

The MIC for each drug can then be provided. The minimum inhibitoryconcentration (MIC) is the minimum test antibiotic concentration towhich the sample is sensitive. An exemplary MIC determination formeropenem based on the results above is depicted in Table 16.

TABLE 16 Mero Mero Mero Mero Inter- 180 [1] 181 [2] 182 [4] 183 [8] 184MIC 185 pretation 186 S 187 S 188 S 189 S 190 <=1 191 S 192 R 193 S 194S 195 S 196 <=2 197 I 198 R 199 R 200 S 201 S 202 <=4 203 I 204 R 205 R206 R 207 S 208 <=8 209 I 210 R 211 R 212 R 213 R 214 >=8 215 R

Example 5. Validation of ABR Assay Utilizing Liquid Growth Medium

Accuracy

Accuracy was assessed by comparing the antibiotic resistance results ofthe test method to a consensus of results obtained by standard referencemethods. A total of 15 isolated organisms, and 20 routinely processedpatient urine samples were tested for resistance to 18 antibiotics, eachtested at multiple concentrations for a total of 57 antibioticconcentrations. Accuracy was assessed regarding Specificity (TrueNegatives), Sensitivity (True Positives), and overall Accuracy (allsamples). The assay showed good accuracy in all three categories (Table17).

TABLE 17 % Accuracy Overall 96% Accuracy Specificity 95% Sensitivity 97%

Precision

Inter-Assay precision was evaluated by testing five samples over threedifferent days. Intra-Assay precision was evaluated by testing the samefive samples in triplicate in a single day. Percent concordance wascalculated to measure the precision of results obtained by this assay.The assay demonstrated very good precision (Table 18).

TABLE 18 Precision Description Intra-Assay Inter-Assay Total # ofMatches 841 1388 Total # of Measurements 855 1425 % Concordance 98% 97%

Analytic

Analytic Sensitivity

Analytic sensitivity was evaluated by creating a dilution series of E.coli and E. faecalis with the lowest bacterial concentration at lessthan 100 cells/mL for each organism. Each dilution level for eachisolate was tested to show reproducibility of results down to the lowestconcentration. 98% correlation was observed across all dilution levelsfor both isolates, indicating the limit of detection (LOD) of this assayis less than 100 cells/ml.

Analytic Specificity

Analytic specificity was evaluated in the context of inhibitory effectof overloading the assay with too many bacterial cells. Lower accuracy(due to false-resistant results) was observed for samples inoculated athigh bacterial concentration. This indicates that all samples must bediluted to the specified cell density post pre-culture and before ABRinoculation.

Pre-Culture Duration Determination

This assay utilizes a pre-culture step prior to introducing samples toantibiotics. The duration of this pre-culture incubation was tested at 6and 16 hours for 2 isolates (E. coli and E. faecalis). Good accuracy foreach isolate was observed after both 6 and 16 hour pre-cultureincubations, indicating a pre-culture window of 6 to 16 hours for thisassay. Results displayed below in Table 19.

TABLE 19 Description # Results % Accuracy Total # of Matches 81 98%Total # of Measurements 83

Incubation

Incubation Duration Determination

Once samples are introduced to antibiotics, they are incubated for 12 to16 hours. This incubation length was determined by obtaining ODmeasurements for Precision samples after 12 and 16 hours of incubation.Good percent concordance was observed for all samples across within a 12to 16 hour incubation window (Table 20).

TABLE 20 Description # Targets % Concordance Total # of Matches 2758 97%Total # of Measurements 2850

Bacterial Growth

Bacterial Growth Confirmation

To confirm turbidity (high OD measurements) are due to bacterial growth,DNA was extracted from wells corresponding to Sensitive and Resistantresults and tested for pathogen identification by PCR. Identificationresults confirm Resistant (turbid) wells contained significantly higherbacterial concentration than Sensitive (clear) wells (Table 21).

TABLE 21 Overall (Cells/mL) Resistant 5,170,897,798 Sensitive 1,341,116Fold-Diff 3,856

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” As used hereinthe terms “about” and “approximately” means within 10 to 15%, preferablywithin 5 to 10%. Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof the invention are approximations, the numerical values set forth inthe specific examples are reported as precisely as possible. Anynumerical value, however, inherently contains certain errors necessarilyresulting from the standard deviation found in their respective testingmeasurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the invention so claimed areinherently or expressly described and enabled herein.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above-citedreferences and printed publications are individually incorporated hereinby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

What is claimed is:
 1. A method for treating a patient having orsuspected of having a polymicrobial infection comprising a combinationof Escherichia coli (E. coli) and Coagulase-negative Staphylococcus(CoNS), the method comprising: a. detecting the presence of both E. coliand CoNS in a source of the infection obtained from the patient; and b.administering to the patient ampicillin, ceftriaxone, tetracycline, orTMP/sulfamethoxazole; wherein E. coli and CoNS together have a decreasedodds of resistance to ampicillin, ceftriaxone, tetracycline, andTMP/sulfamethoxazole, thus ampicillin, ceftriaxone, tetracycline, andTMP/sulfamethoxazole are effective for killing or inhibiting growth ofE. coli and CoNS to treat the polymicrobial infection.
 2. The method ofclaim 1, wherein E. coli and CoNS are detected in the source of theinfection without first being isolated.